NMR-Based Metabolomics of Rat Hippocampus, Serum, and Urine in Two Models of Autism.

Autism spectrum disorders (ASDs) are increasingly diagnosed as developmental disabilities of unclear etiology related to genetic, epigenetic, or environmental factors. The diagnosis of ASD in children is based on the recognition of typical behavioral symptoms, while no reliable biomarkers are available. Rats in whom ASD-like symptoms are due to maternal administration of the teratogenic drugs valproate or thalidomide on critical day 11 of pregnancy are widely used models in autism research. The present studies, aimed at detecting changes in the levels of hydrophilic and hydrophobic metabolites, were carried out on 1-month-old rats belonging to the abovementioned two ASD models and on a control group. Analysis of both hydrophilic and hydrophobic metabolite levels gives a broader view of possible mechanisms involved in the pathogenesis of autism. Hippocampal proton magnetic resonance (MRS) spectroscopy and ex vivo nuclear magnetic resonance (NMR) analysis of serum and urine samples were used. The results were analyzed using advanced statistical tests. Both the results of our present MRS studies of the hippocampus and of the NMR studies of body fluids in both ASD models, particularly from the THAL model, appeared to be consistent with previously published NMR results of hippocampal homogenates and data from the literature on autistic children. We detected symptoms of disturbances in neurotransmitter metabolism, energy deficit, and oxidative stress, as well as intestinal malfunction, which shed light on the pathogenesis of ASD and could be used for diagnostic purposes. These results confirm the usefulness of the noninvasive techniques used in ASD studies.

Hippocampal Metabolite Profiles in Two Rat Models of Autism: NMR-Based Metabolomics Studies.

Autism spectrum disorders (ASDs) are increasingly being diagnosed. Hypotheses link ASD to genetic, epigenetic, or environmental factors. The role of oxidative stress and the imbalance between excitatory and inhibitory neurotransmission in the pathogenesis of ASD has been suggested. Rats in which ASD symptoms are induced by valproate (VPA) or thalidomide (THAL) application in utero are useful models in ASD studies. Our study investigated whether rats in ASD models show changes in metabolite levels in the brain consistent with the hypothetical pathomechanisms of ASD. Female rats were fed one dose of 800 mg/kg VPA or 500 mg/kg THAL orally on the 11th day of gestation, and 1-month offspring were used for the experiments. Metabolic profiles from proton nuclear magnetic resonance spectroscopy of hydrophilic and hydrophobic extracts of rat hippocampi were subjected to OPLS-DA statistical analysis. Large differences between both models in the content of several metabolites in the rat hippocampus were noticed. The following metabolic pathways were identified as being disturbed in both ASD models: steroid hormone biosynthesis; fatty acid biosynthesis; the synthesis and degradation of ketone bodies; glycerophospholipid metabolism; cholesterol metabolism; purine metabolism; arginine and proline metabolism; valine, leucine, and isoleucine biosynthesis and degradation. These results indicate disorders of energy metabolism, altered structure of cell membranes, changes in neurotransmission, and the induction of oxidative stress in the hippocampus. Our data, consistent with hypotheses of ASD pathomechanisms, may be useful in future ASD studies, especially for the interpretation of the results of metabolomics analysis of body fluids in rat ASD models.

NMDA receptor antagonism does not inhibit induction of ischemic tolerance in gerbil brain in vivo.

Effects of high and moderate affinity uncompetitive NMDA receptor antagonists (+)MK-801 and memantine on ischemic tolerance were compared in relation to telemetrically controlled brain temperature. The tolerance to an injurious 3 min test of global forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Normothermic preconditioning was virtually harmless, and greatly reduced neurodegeneration evoked by test ischemia. In hyperthermic animals it was injurious and failed to induce tolerance. Memantine (5 mg/kg) and (+)MK-801 (3 mg/kg) injected i.p. 1 h before preconditioning did not inhibit ischemic tolerance in the normothermic gerbils, while in hyperthermic animals treated with (+)MK-801 ischemic tolerance was partially restored. Subchronic 3 day infusion of memantine (30 mg/kg/day) significantly decreased neurodegeneration, and preconditioning in the normothermic gerbils further reduced neuronal damage. Hyperthermia exacerbated preconditioning ischemia and in this way reduced expression of tolerance, while (+)MK-801 partially reversed this effect. Our results do not confirm previous reports on the role of NMDA receptors in the induction of ischemic tolerance in gerbils.

Activation of N-methyl-D-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells.

In cultured granule cells prelabeled with [3H]arachidonate the activation of excitatory amino acid receptors by various agonists results in a dose-dependent stimulation of [3H]arachidonic acid release. Glutamate and aspartate were the most potent agonists, whereas N-methyl-D-aspartate, kainate and quisqualate were less potent. Other neurotransmitter receptor agonists--GABA, baclofen and norepinephrine--were inactive, while carbachol induced only a slight effect. Since the transmitter-mediated release of [3H]arachidonate was blocked by phencyclidine, a selective inhibitor of NMDA-sensitive glutamate receptors, it can be inferred that the effects of all other receptor agonists were indirectly mediated via the release of glutamate from granule cells. Aspartate-evoked release was Ca2+-dependent and was abolished by the glutamate receptor inhibitors: Mg2+ ions and 2-amino-5-phosphonovalerate. The inhibitors of phospholipase A2, quinacrine and p-bromophenacyl bromide, decreased the release of [3H]arachidonate in a dose-related manner.

Glycine and D-serine act as positive modulators of signal transduction at N-methyl-D-aspartate sensitive glutamate receptors in cultured cerebellar granule cells.

In cultures of rat neonatal cerebellar granule cells the signal transduction at ionotropic NMDA-sensitive glutamate receptors (GC1) was measured as an increase of influx of 45Ca2+. This transmitter-mediated influx of Ca2+ was enhanced by glycine and D-serine in a dose-dependent manner. D-Alanine was less active than glycine and D-serine, while L-alanine and L-serine were inactive. These amino acids failed to activate basal influx of Ca2+. Activation of calcium influx at GC2 receptors by kainate was unchanged by the amino acids mentioned above. Glycine and D-serine increased the potency but failed to change the efficacy of GC1 agonists. This action was not changed by strychnine. The enhancement of aspartate signal transduction by glycine and D-serine was inhibited by the noncompetitive GC1 receptor antagonist, phencyclidine, but was even more evident in presence of Mg2+ ions. Hence, glycine and D-serine may function as positive allosteric modulators of signal transduction at NMDA-sensitive (GC1) glutamate receptors.

Effects of taurine on calcium binding and accumulation in rabbit hippocampal and cortical synaptosomes.

The effect of taurine on Ca(2+) binding and uptake was studied with rabbit brain cortical and hippocampal synaptosomes. Taurine (25 mM) increased by 25% the high affinity (45)Ca(2+) binding in the cortical fraction and by 55% in hippocampal synaptosomes but had no effect on low affinity Ca(2+) binding. Taurine decreased significantly the fluorescence of the chlorotetracycline-hydrophobic Ca(2+) chelate probe in both synaptosomal fractions which suggests a shift of bound Ca(2+) from the hydrophobic to the hydrophilic part of the membranes. The uptake of (45)Ca(2+) by rabbit brain synaptosomes, when measured in control and 65 mK K(+)-containing media, was not influenced by taurine. However, taurine inhibited significantly the (45)Ca(2+) uptake in synaptosomes incubated in media containing moderately increased K(+) concentrations (14 and 20 mM K(+)). The effects of taurine are discussed in conjunction with its stabilizing effect on excitable membranes.

Arachidonic acid release from K(+)-evoked depolarization of brain synaptosomes.

Rat brain synaptosomes prelabeled with [(14)C]arachidonate in their phospholipids were superfused with well oxygenated Krebs-Ringer-bicarbonate solution containing 0.2% BSA and subsequently depolarized by elevating the K(+) concentration in the superfusion medium from 5 to 55 mM. The efflux of labeled arachidonate at steady state was 0.19% (n = 12) of total radioactivity per min. In the presence of 2.5 mM Ca(2+), high K(+) (55 mM) in the medium elicited an increase in arachidonate efflux which amounted to 121.4% (n = 6) of control. Both Ca(2+) and BSA were required for the stimulated efflux of arachidonate during K(+)-depolarization. Under the same condition, K(+)-stimulation also evoked the release of [(3)H]norepinephrine which was preloaded into the synaptosomes prior to superfusion. EGTA abolished the stimulated release of both arachidonate and norepinephrine during K(+)-depolarization. These results, together with the loss of labeled arachidonic acid from phospholipids (Majewska and Sun, 1982), indicate that deacylation of membrane lipids is involved in synaptic functions.

NMDA-induced 45Ca release in the dentate gyrus of newborn rats: in vivo microdialysis study.

This in vivo study, aimed at detecting the N-methyl-D-aspartate (NMDA) evoked Ca(2+)-induced Ca(2+) release from intracellular stores in the neonatal rat brain, demonstrates that the application of 5 mM N-methyl-D-aspartate via a microdialysis probe for 20 min to the dentate gyrus (DG) of halotane-anesthetized 7 day-old (postnatal day 7, PND 7) rats induces a prolonged decrease in Ca(2+) concentration in an initially calcium-free dialysis medium, indicative of a drop in the extracellular concentration of Ca(2+) and Ca(2+) influx to neurons. In parallel experiments, a huge NMDA-evoked release of 45Ca from the pre-labeled endogenous Ca(2+) pool was observed and interpreted as the expression of intracellular Ca(2+) release. Dantrolene (100 microM) significantly inhibited the NMDA-induced 45Ca release, whereas 250 microM ryanodine exerted an unspecific biphasic effect. Autoradiographic and immunocytochemical detection of ryanodine receptors and calbindin D(28K), respectively, in the hippocampal region of PND 7 rats displayed a pronounced expression of [3H]ryanodine binding sites in the DG, but only a slight immunoreactivity of calbindin D(28K). Plastic changes in neurons or excitotoxic neuronal damage induced by the activation of NMDA receptors are mediated by Ca(2+) signals, resulting from an influx of extracellular Ca(2+), and also in some neurons, from the release of intracellular Ca(2+). Our previous in vivo microdialysis experiments visualized NMDA-evoked 45Ca release in the adult rat dentate gyrus, attributable to Ca(2+)-induced Ca(2+) release from the ryanodine-sensitive pool. An additional role of calbindin in the mechanism of this phenomenon has been suggested. This aspect has not been studied in vivo in newborn rats. Our present results indicate that the release of 45Ca from the prelabeled intracellular, dantrolene-sensitive Ca(2+) pool in the DG neurons of immature rats, most probably representing a phenomenon of Ca(2+)-induced Ca(2+) release, significantly participates in the generation of NMDA receptor-mediated intracellular Ca(2+) signals, whereas the role of calbindin D(28K) in the mechanism of 45Ca release is negligible.

Extracellular calcium in the hippocampus of unanesthetized rabbits monitored with dialysis-perfusion.

Extracellular levels of Ca2+ in the rabbit hippocampus were investigated in vivo with perfusion-dialysis. A thin semipermeable tubing was implanted and perfused at a constant rate with a Ca2+-free medium. Ca2+ levels in the dialysate were measured with an ion-specific electrode or as radioactivity after labeling of the endogenous pool with 45CaCl2. The system was characterized with model studies. The usefulness of the method is demonstrated for studies of, for example chemically evoked changes in Ca2+ concentrations extracellularly in CNS regions of non-anesthetized, freely moving rabbits. Moreover, measurements of Ca2+ changes are easily correlated with neurotransmitter levels in the same samples.

Species differences in cerebral taurine concentrations correlate with brain water content.

The notion that taurine (Tau) has an osmoregulatory function in the mammalian brain has not been established, although it has been reported that the severity of hyponatremic edema is proportional to cerebral [Tau]. Tau pools are not easily altered in vivo, but the fact that there are large differences in cerebral taurine levels between mice, rats and guinea pigs offers an opportunity to determine whether endogenous Tau affects volume regulation of the brain in hyposmolal conditions. This issue was investigated by injecting saline or distilled water intraperitoneally at 150 ml/kg in anesthetized mice, rats and guinea pigs. The animals were decapitated 4 h later, and blood osmolality, cortical specific gravity, Na+, K+ and amino acid concentrations were determined. In controls, blood osmolality and specific gravity of the cortex were highest in the mouse (304 +/- 3 mmol/kg; 1.0488 +/- 0.0003 kg/l), followed by the rat (294 +/- 1; 1.0462 +/- 0.0002) and the guinea pig (285 +/- 2; 1.0445 +/- 0.0002). There was a correlation between these measures and cortical Tau levels which were 10.31 +/- 0.36 mmol/kg in mouse cortex, 6.31 +/- 0.18 in rat cortex and 1.37 +/- 0.06 in guinea pig cortex. Despite these differences, water-induced cerebrocortical swelling did not differ between the species studied. Interspecies variation in cortical osmolality did not relate to [Na+] and [K+], since the levels of these electrolytes were higher in the guinea pig cortex than in the rat and mouse cortex. After administration of water, the levels of Na+ and K+ were reduced in rat and guinea pig cortex, while only [Na+] was significantly decreased in mouse cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of N-methyl-D-aspartate on Ca2+ homeostasis in developing and adult rat striatum: in vivo microdialysis approach.

This in vivo study concerns developmental differences in the sensitivity of striatal neurons to N-methyl-D-aspartate (NMDA). Changes in calcium homeostasis in adult vs immature rats at postnatal days 8-10, evoked by NMDA, were evaluated by measurements of 45Ca efflux and of Ca2+ taurine and phosphoethanolamine concentrations in striatal microdialysates. The efflux of [14C]sucrose was employed in order to measure changes in extracellular space volume. In adult rats the addition of 5 mM NMDA for 20 min to the perfusion medium resulted in a 30-40% increase in 45Ca efflux, and in a 15% increase in [14C]sucrose efflux. Ten minutes after NMDA perfusion, 45Ca and [14C] sucrose efflux returned to the baseline. No significant changes in Ca2+ or amino acid concentrations were observed in the dialysate of the adult rat striatum. NMDA perfusion in the striatum of immature rats initially induced a transient (5 min) increase in the efflux of 45Ca (by 13%) and [14C]sucrose (by 9%). This was followed by a prolonged (lasting 45-50 min) 45% decrease in 45Ca efflux, an 80% reduction of Ca2+ concentration, and increases in taurine and phosphoethanolamine concentrations in the dialysate, whereas [14C]sucrose efflux recovered within 10 min. These data illustrate differences in the NMDA response between developing and adult rat striatum. Only in developing rats did NMDA induce a large and prolonged influx of extracellular calcium to neurons that may explain the enhanced NMDA neurotoxicity in immature rats.

Subcellular distribution of calcium and ultrastructural changes after cerebral hypoxia-ischemia in immature rats.

Recent data imply that mitochondrial regulation of calcium is critical in the process leading to hypoxic-ischemic brain injury. The aim was to study the subcellular distribution of calcium in correlation with ultrastructural changes after hypoxia-ischemia in neonatal rats. Seven-day-old rats were subjected to permanent unilateral carotid artery ligation and exposure to hypoxia (7.7% oxygen in nitrogen) for 90 min. Animals were perfusion-fixed after 30 min, 3 h or 24 h of reperfusion. Sections were sampled for light microscopy and electron microscopy combined with the oxalate-pyroantimonate technique. At 30 min and 3 h of reflow, a progressive accumulation of calcium was detected in the endoplasmic reticulum, cytoplasm, nucleus and, most markedly, in the mitochondrial matrix of neurons in the gray matter in the core area of injury. Some mitochondria developed a considerable degree of swelling reaching a diameter of several microm at 3 h of reflow whereas the majority of mitochondria appeared moderately affected. Chromatin condensation was observed in nuclei of many cells with severely swollen mitochondria with calcium deposits. A whole spectrum of morphological features ranging from necrosis to apoptosis was seen in degenerating cells. After 24 h, there was extensive injury in the cerebral cortex as judged by breaks of mitochondrial and plasma membranes, and a general decrease of cellular electron density. In the white matter of the core area of injury, the axonal elements exhibited varicosity-like swellings filled with calcium-pyroantimonate deposits. Furthermore, the thin myelin sheaths were loaded with calcium. Numerous oligodendroglia-like cells displayed apoptotic morphology with shrunken cytoplasm and chromatin condensation, whereas astroglial necrosis was not seen. In conclusion, markedly swollen 'giant' mitochondria with large amounts of calcium were found at 3 h of reperfusion often in neuronal cells with condensation of the nuclear chromatin. The results are discussed in relation to mitochondrial permeability transition and activation of apoptotic processes.

Acute hepatic encephalopathy decreases potassium-evoked calcium uptake in astrocytes but not in synaptosomes of the rat.

45CaCl2 uptake at low (5 mM), and high, depolarizing (65 mM) KCl concentration was measured in a fraction enriched in astrocytes, a crude mitochondrial-synaptosomal (P2) preparation and in purified synaptosomes derived from normal rats and from rats with thioacetamide-induced acute hepatic encephalopathy (HE). HE was found to reduce the potassium-evoked component of the astroglial uptake to 50% of its control level, without affecting the uptake into the P2 fraction or synaptosomes. The results are in keeping with the view that astrocytes are the cells whose metabolism and functions are predominantly affected during HE.

Pattern of neuronal loss in the rat hippocampus following experimental cardiac arrest-induced ischemia.

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, dissector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P<0.05). At this time, no neuronal loss was observed in other cornu Ammonis sectors or the granular layer of the dentate gyrus. On the 14th postischemic day, further neuronal loss in the sector CA1 pyramidal layer was noticed. At this time, this sector contained 31% fewer pyramidal neurons than on the third day (P<0.05) and 58% fewer than in the control group (P<0.01). On the 14th day, neuronal loss in other hippocampal subdivisions also was observed. The pyramidal layer of sector CA3 contained 36% fewer neurons than in the control group (P<0.05), whereas the granular layer of the dentate gyrus contained 40% fewer (P<0.05). The total number of pyramidal neurons in sector CA2 remained unchanged. After the 14th day, no significant alterations in the total number of neurons were observed in any subdivision of the hippocampus until the 12th month of observation. Unbiased morphometric analysis emphasizes the exceptional susceptibility of sector CA1 pyramidal neurons to hypoxia/ischemia but also demonstrates significant neuronal loss in sector CA3 and the dentate granular layer, previously considered 'relatively resistant'. The different timing of neuronal dropout in sectors CA1 and CA3 and the dentate gyrus may implicate the existence of region-related properties, which determine earlier or later reactions to ischemia. However, the hippocampus has a unique, unidirectional system of intrinsic connections, whereby the majority of dentate granular neuron projections target the sector CA3 pyramidal neurons, which in turn project mostly to sector CA1. As a result, the early neuronal dropout in sector CA1 may result in retrograde transynaptic degeneration of neurons in other areas. The lack of neuronal loss in sector CA2 can be explained by the resistance of this sector to ischemia/hypoxia and the fact that this sector is not included in the major chain of intrahippocampal connections and hence is not affected by retrograde changes.

Differences between rats and rabbits in NMDA receptor-mediated calcium signalling in hippocampal neurones.

In vivo microdialysis combined with the measurement of (45)Ca(2+) efflux from prelabelled hippocampus demonstrated a pronounced N-methyl-D-aspartate (NMDA)-evoked (45)Ca(2+) release to the dialysate in the rat dentate gyrus (DG) and CA1, whereas in rabbit a slight release of (45)Ca(2+) was observed only in the DG. In vitro, we noticed that the NMDA-evoked increase in Fura-2 detected intracellular Ca(2+) concentration in synaptoneurosomes from the rat, but not from the rabbit hippocampus, was strongly inhibited by the ryanodine receptor (RyR) antagonists dantrolene and ryanodine. To establish the mechanism of these differences, we characterised their possible dependence on the expression of RyR and their co-localisation with the calcium binding protein calbindin D(28k). A pronounced expression of [(3)H]ryanodine binding sites in the rat DG, which is only slight in the CA1, was demonstrated whereas in rabbit they were only found in the DG. The pattern of expression of calbindin D(28k) immunoreactivity and RyR in the rat and rabbit hippocampus was similar. These results suggest that the functional role of RyR in the generation of the NMDA receptor-mediated intracellular Ca(2+) signalling in the rabbit hippocampal neurones is marginal when compared to the rat. These differences reflect a diverse expression of RyR in both species. The corresponding differences in calbindin D(28k) immunoreactivity are most probably secondary in nature.

Induction of permeability transition and swelling of rat brain mitochondria by glutamine.

Exposure of rat cerebral mitochondria to 2-10 mM glutamine (Gln) for 20 min, produced a concentration-dependent, gradual decrease of light scattering reflecting mitochondrial swelling. The light scattering decreasing effect of 5 mM Gln was attenuated by 0.5 microM cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition (mPT) induction. Histidine (His), which is a potent inhibitor of high affinity Gln uptake to mitochondria, attenuated Gln-induced decrease of mitochondrial light scattering when added at equimolar concentration, and abolished the decrease when added at 15 mM concentration shortly before addition of Gln. His inhibited the uptake of 5 mM [14C]Gln in a concentration-dependent manner as measured during 3 min incubation. CsA did neither affect [14C]Gln uptake nor modified its inhibition by His. The effects of 5 mM His and 0.5 microM CsA on mitochondrial light scattering were additive, indicating that mitochondrial swelling represents a cumulative effect of Gln -driven entry of osmotically obligated water and induction of mPT. Addition of ammonium ions at neurotoxic concentrations neither influenced the decrease of light scattering induced by Gln, nor produced any change in light scattering when added alone. The results point to mitochondrial swelling and subsequent activation of mPT, as one of the potential mechanisms by which Gln induces metabolic disturbances in the brain in hyperammonemic conditions.

Ischemia modifies protein distribution in gerbil brain subcellular fractions.

This work deals with changes in sedimentation of proteins in subcellular fractions isolated from the ischemic brain of Mongolian gerbils. We analysed protein content and the activity of marker enzymes in P2, S2, M and S3 subfractions isolated after 10 min ischemia, ischemia combined with apnea or 1 h later, after recirculation. On the basis of protein balance, the distribution of marker enzymes and electrophoretic analysis of proteins in brain subfractions it was shown that in ischemia some fraction of proteins which are normally cytosolic and microsomal, cosediment with the crude mitochondrial pellet. This effect, aggravated in ischemia complicated by apnea, is reversible and after 1 h recirculation the protein distribution in the brain normalizes completely.

Early changes in extracellular amino acids and calcium concentrations in rabbit hippocampus following complete 15-min cerebral ischemia.

The effect of cerebral ischemia on extracellular amino acids and calcium content and on the permeability of the blood-brain barrier was studied by in vivo dialysis of rabbit hippocampus. This was combined with physiological and neurophysiological measurements. It was found that immediately after 15-min ischemia extracellular concentrations of glutamate, aspartate and taurine increased 3-, 2- and 6-fold, respectively, whereas a maximal, 7-fold increase of phosphoethanolamine and persistent elevation of glutamate were observed 45 min after ischemia. Extracellular calcium concentration, monitored with 45Ca2+, increased by 10% during the initial phase of ischemia, and decreased to approx. 74% of the basal level 10 min after ischemia. Recovery of extracellular calcium content was not attained until 45 min of recirculation, at which time the first signs of return of bioelectric activity were noted. Increased permeability of the blood-brain barrier to fluoresceine developed immediately after ischemia and persisted up to 2 h of recirculation. The obtained results are discussed in reference to the noted simultaneity of changes in extracellular excitatory amino acids and calcium concentrations and of brain bioelectric activity during and after ischemia. Causal relations between these effects are suggested.

Changes of intracellular calcium homeostasis in brain cortical structures during anoxia in vivo and in vitro.

This work characterizes the anoxia-evoked changes in the content of bound calcium (Cab) in brain cortex membraneous structures, studied in vivo, on living brain cortex preparation and in vitro, on subcellular fractions (synaptosomes, microsomes and mitochondria) in anoxic conditions. The chlorotetracycline (CTC) fluorescent chelate probe was used to monitor changes of Cab content in hydrophobic domains of intracellular membranes. In in vivo experiments the bioelectric activity of single neurons was recorded simultaneously with measurements of Ca-CTC fluorescence. In vitro experiments were supplemented with determinations of synaptosomal 45Ca-uptake. It was found that the response of cortical neurons to anoxia is manifested in a decrease of a portion of Ca2+ bound with membrane hydrophobic domains. These in vivo changes preceded the noticeable disturbances of neuronal electric activity. An anoxia-evoked drop in Cab was also clearly demonstrated in vitro, irrespective of K+ (for synaptosomes) or Na+ (for mitochondria) concentrations in incubation media, although the additional effect of Cab displacement was noted when Na+/K+ concentrations were modified in order to simulate their changes occurring in anoxic conditions. It was found that the membranes of different neuronal compartments are not uniformly vulnerable to anoxia in vitro as the anoxic decrease in Cab content occurred in synaptosomes and microsomes much sooner than in mitochondria. Therefore, in vivo and in vitro experiments visualized high sensitivity of Ca2+-binding mechanisms in different neuronal membranes to anoxia. The anoxia-evoked displacement of a portion of Cab to the free ionic form may trigger a complex intracellular response determining anoxic reactions and post-anoxic recovery.

Responses to reversible anoxia of intracellular free and bound Ca(2+) in rat cortical slices.

Severe anoxia induces destabilisation of intracellular calcium homeostasis in neurones. The mechanism of this effect, and particularly the interrelationship between changes in intracellular concentration of free Ca(2+) ions and the content of the intracellular Ca(2+) stores, during and after anoxia, is not clear. We used a superfusion system of rat olfactory cortical slices for the fluorimetric estimation of changes in the intracellular concentration of free Ca(2+) ions and in the level of bound Ca(2+), utilising the fluorescent indicators Fura-2 and chlortetracycline, respectively. It was found that 10-min normoglycaemic anoxia results in simultaneous decrease in bound and increase in free Ca(2+) levels, whereas during 60-min reoxygenation, we detected an increase in both indices. The NMDA receptor antagonists MK-801 and APV attenuated changes in free Ca(2+) level during anoxia and reoxygenation and intensified anoxia-evoked decrease in bound Ca(2+) content, whereas a late post-anoxic increase in bound Ca(2+) was abolished. These data suggest that the influx of extracellular Ca(2+) to neurones via NMDA receptors, plays a critical role in the rise of intracellular free Ca(2+) concentration during and after anoxia. Biphasic changes in bound Ca(2+) content during anoxia and reoxygenation may reflect an anoxia-induced release of Ca(2+) from intracellular stores, followed later by a neuronal calcium overload and refilling of intracellular Ca(2+) binding sites.