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.

Bastadin 12 and ryanodine reveal similarities between thapsigargin- and tetrabromobisphenol A-induced intracellular Ca(2+) release in cultured cerebellar granule cells.

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant with recognized neuro- and cytotoxic properties that are presumably mediated by intracellular Ca(2+) release. Other studies have demonstrated that ryanodine is able to inhibit Ca(2+) efflux from skeletal sarcoplasmic reticulum (SR) membranes in response to the known Ca(2+) releaser thapsigargin, provided that the macrocyclic brominated tyrosine derivative bastadin 5 is also present. Similar effects supporting the role of ryanodine receptors in thapsigargin-evoked Ca(2+) release have been observed in primary cultures of rat cerebellar granule cells (CGCs). Here, we used CGCs and the fluorescent intracellular Ca(2+) probe fluo-3 to test the following hypotheses: (1) TBBPA shares Ca(2+) releasing properties with thapsigargin, and (2) synthetic bastadin 12 can replace bastadin 5 as a pharmacological tool to identify these similarities. The results demonstrated that either 200 nM thapsigargin or 30 µM bastadin 12 alone induced an increase in the intracellular Ca(2+) level in CGCs, whereas 2.5 and 10 µM bastadin 12 had no effect on the basal Ca(2+) concentration. The thapsigargin-induced Ca(2+) release was partially reduced by co-administration of either 2.5 µM bastadin 12 or 200 µM ryanodine, and the release of Ca(2+) was nearly completely attenuated by these compounds when they were given together. TBBPA (5, 10 and 25 µM) administration caused a concentration-dependent increase in CGC Ca(2+) levels. Administration of 2.5 µM bastadin 12 with 200 µM ryanodine blocked the increase in intracellular Ca(2+) evoked by 10 µM TBBPA, although these compounds were ineffective when applied separately. These results indicate that bastadin 12 may replace bastadin 5 when testing the ability of ryanodine to inhibit Ca(2+) release from the intracellular stores of cultured neurons, and our findings support the hypothesis that TBBPA and thapsigargin induce intracellular Ca(2+) release through a common mechanism.

Low molecular weight thiols reduce thimerosal neurotoxicity in vitro: modulation by proteins.

Thimerosal (TH), an ethylmercury complex of thiosalicylic acid has been used as preservative in vaccines. In vitro neurotoxicity of TH at high nM concentrations has been reported. Although a number of toxicological experiments demonstrated high affinity of mercury to thiol groups of the extracellular amino acids and proteins that may decrease concentration of free TH in the organism, less is known about the role of interactions between proteins and amino acids in protection against TH neurotoxicity. In the present study we examined whether the presence of serum proteins and of l-cysteine (Cys), d,l-homocysteine (Hcy), N-acetyl cysteine (NAC), l-methionine (Met) and glutathione (GSH) in the incubation medium affects the TH-induced changes in the viability, the intracellular levels of calcium and zinc and mitochondrial membrane potential in primary cultures of rat cerebellar granule cells. The cells were exposed to 500 nM TH for 48 h or to 15-25 µM TH for 10 min. Our results demonstrated a decrease in the cells viability evoked by TH, which could be prevented partially by serum proteins, albumin or in a dose-dependent manner by 60, 120 or 600 µM Cys, Hcy, NAC and GSH, but not by Met. This neuroprotection was less pronounced in the presence of proteins. Incubation of neurons with TH also induced the rise in the intracellular calcium and zinc concentration and decrease in mitochondrial membrane potential, and these effects were abolished by all the sulfur containing compounds studied and administered at 600 µM concentration, except Met. The loss of the ethylmercury moiety from TH as a result of interaction with thiols studied was monitored by (1)H NMR spectroscopy. This extracellular process may be responsible for the neuroprotection seen in the cerebellar cell cultures, but also provides a molecular pathway for redistribution of TH-derived toxic ethylmercury in the organism. In conclusion, these results confirmed that proteins and sulfur-containing amino acids applied separately reduce TH neurotoxicity, while their combination modulates in more complex way neuronal survival in the presence of TH.

Differential changes in phosphorylation of tau at PHF-1 and 12E8 epitopes during brain ischemia and reperfusion in gerbils.

Cortical neurons are vulnerable to ischemic insult, which may cause cytoskeletal changes and neurodegeneration. Tau is a microtubule-associated protein expressed in neuronal and glial cells. We examined the phosphorylation status of tau protein in the gerbil brain cortex during 5 min ischemia induced by bilateral common carotid artery occlusion followed by reperfusion for 20 min to 7 days. Control brain homogenates contained 63, 65 and 68 kD polypeptides of tau immunoreactive with Alz 50, Tau 14 and Tau 46 antibodies raised against non-phosphorylated tau epitopes. Gerbil tau was also immunoreactive with some (PHF-1 and 12E8) but not all (AT8, AT100, AT180 and AT270) antibodies raised against phosphorylated tau epitopes. PHF-1 recognized a single 68 kD polypeptide and 12E8 bound the 63 kD polypeptide. During 5 min ischemia, PHF-1 immunoreactivity declined to 6%, then recovered to control levels after 20 min of blood recirculation and subsequently increased above control values 3 and 7 days later. In contrast, 12E8 immunoreactivity remained stable during ischemia and reperfusion. Our results suggest that the two phosphorylated epitopes of tau are regulated by different mechanisms and may play different roles in microtubule dynamics. They may also define various pools of neuronal/glial cells vulnerable to ischemia.

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.

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.

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.

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.

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.

NMDA receptors and nitric oxide regulate prostaglandin D2 synthesis in the rabbit hippocampus in vivo.

The aim of this in vivo microdialysis study was to characterise the regulation of prostaglandin D2 (PgD2) synthesis by NMDA receptors in the rabbit hippocampus in relation to changes in extracellular Ca2+ concentration ([Ca2+]e) and nitric oxide (NO) levels. Samples of dialysate were analysed for changes in PgD2 concentration, in [Ca2+]e and in the level of NO. The results demonstrated that a 20-min pulse application of 0.1-2.5 mM NMDA via a microdialysis probe induced a prolonged stimulation of PgD2 release that was sensitive to competitive NMDA receptor antagonists. An inhibitor of voltage-sensitive Na+ channels, tetrodotoxin, did not influence this effect but significantly suppressed basal PgD2 production, whereas a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), prevented NMDA-evoked NO release and inhibited NMDA-induced PgD2 release in an L-arginine-sensitive manner. NO donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, stimulated PgD2 release. NMDA-evoked decrease in [Ca2+]e was insensitive to TTX and L-NAME. These results demonstrate an in vivo NMDA receptor-mediated modulation of PgD2 synthesis in the brain, in which NO participates.

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.

In vivo brain microdialysis as a tool in studies of neuroprotective effects of cyclosporin A in acute excitotoxicity.

The mitochondrial permeability transition (MPT) resulting from calcium-induced opening of cyclosporin A (CsA)-sensitive megachannels, leading to deenergisation of mitochondria and release of pro-apoptotic cytochrome c, has been implicated in the pathomechanism of excitotoxic neurodegeneration. The aim of this work was to test neuroprotective potential of CsA in the model of N-methyl-D-aspartate-(NMDA)-induced excitotoxicity in vivo, and to verify utility of microdialysis of the rabbit hippocampus in vivo for these mechanistic studies. In vitro experiments demonstrated that the early rapid phase of Ca(2+)-induced swelling of isolated brain mitochondria, and of accompanying cytochrome c release, was strongly inhibited by 0.5 microM CsA. In the in vivo experiments 1 mM NMDA was applied for 20 min to the hippocampus in a control, or 5 microM CsA-containing dialysis medium via transhippocampal microdialysis probes, and changes in extracellular Ca2+ concentration and in NO release were monitored. Application of NMDA induced a prolonged decrease in the extracellular concentration of Ca2+, reflecting influx of Ca2+ to stimulated neurones. CsA only slightly enhanced this effect. NMDA induced also release of NO to the dialysis medium. Morphological examination 30 min after NMDA application visualised swelling of dendritic mitochondria and cisternae of endoplasmatic reticulum of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of microdialysis probes. CsA prevented mitochondrial swelling. After 24 h degeneration of the CA1 pyramidal neurones close to a microdialysis probes was observed, which was partially prevented in CsA-treated rabbits. These results indicate that the mechanism of CsA nuroprotection may be at least in part ascribed to prevention of MPT. Microdialysis of the rabbit hippocampus combined with NMDA excitotoxicity appeared to be useful in mechanistic studies of CsA neuroprotection.

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.

Role of Ca2+ release from ryanodine stores in generation of NMDA-induced Ca2+ signal in rabbit hippocampus.

In vivo microdialysis combined with measurements of 45Ca efflux from pre-labelled rat hippocampus has been utilised in our laboratory to demonstrate NMDA-evoked 45Ca2+ release to dialysate, reflecting calcium-induced calcium release (CICR) via ryanodine receptors (RyR). In the present study we attempted to reproduce this phenomenon in the rabbit hippocampus. Application of 1 mM NMDA to dialysis medium induced a decrease in Ca2+ concentration in dialysate, as a result of extracellular Ca2+ influx to neurones. The release of 45Ca2+ was not observed, instead a decrease in 45Ca2+ efflux rate from the NMDA treated rabbit hippocampus was noted, along with release to dialysate of prostaglandin D2, taurine and phosphoethanolamine. All these effects, reflecting different steps of intracellular calcium signalling, were insensitive to 100 microM dantrolene and 50 microM ryanodine, RyR modulators known to interfere with NMDA-evoked 45Ca2+ release in the rat hippocampus. Thus, although the results of this study demonstrate the role of extracellular Ca2+ influx to neurones in NMDA-evoked generation of Ca2+ signal in the rabbit hippocampus, the activity of CICR was not detected.

N-methyl-D-aspartate receptor-mediated, calcium-induced calcium release in rat dentate gyrus/CA4 in vivo.

Previously, by using in vivo microdialysis, we demonstrated a huge release of 45Ca2+ from prelabeled tissues to dialysate that was evoked by application of N-methyl-D-aspartate (NMDA) to the rat dentate gyrus (DG) and sector 4 of the cornu ammonis. To establish the mechanism of this phenomenon, in the present study, we characterized its NMDA receptor dependence, investigated the mechanism of 45Ca2+ removal from the cells, and evaluated the possible involvement of calcium-binding protein calbindin D28k and of ryanodine receptors. Microdialysis experiments demonstrated a dose-response relation between NMDA and 45Ca2+ release and sensitivity of this phenomenon to inhibition by 10 microM MK-801 and 5 mM 5-(N,N-dimethyl)-amiloride, thus indicating the NMDA receptor dependence and a role of Na+/Ca2+ exchanger in mediating 45Ca2+ release from cells. Immunocytochemical experiments confirmed that DG granule cells in the investigated inbred rat strain are strongly calbindin D28k-immunopositive, indicating probable involvement of this protein. However, microdialysis studies demonstrated that NMDA-evoked 45Ca2+ release was suppressed by 100 microM dantrolene and 250 microM ryanodine, whereas 50 microM ryanodine stimulated this effect. This points to a key role in the investigated phenomenon of calcium-induced calcium release (CICR) via ryanodine receptors. To our knowledge, this is the first in vivo demonstration of NMDA-evoked CICR. We postulate the usefulness of microdialysis in such studies.

Effects of caffeine on NMDA-evoked 45Ca2+ release in the rate dentate gyrus in vivo.

Caffeine in 10(-2) M concentration per se activates ryanodine receptors (RyR) in vitro, thereby increasing the intracellular concentration of Ca2+ ([Ca2+]i). In general opinion, caffeine applied in vivo in much lower doses does not affect [Ca2+]i in neurones. However, it was recently demonstrated that caffeine in low concentrations in vitro potentiates evoked Ca2+ release in neurones via RyR. Microdialysis of the rat dentate gyrus (DG), combined with measurement of 45Ca2+ efflux, has been used in our laboratory to study in vivo NMDA-evoked calcium induced calcium release (CICR) via RyR. The aim of the present microdialysis study was to investigate in vivo effects of caffeine, applied systemically in a pharmacologically-relevant dose, and locally in the dialysis medium in very high concentration, on the NMDA-evoked CICR in DG neurones. To ensure steady brain concentration of caffeine, its systemic (i.p.) administration in a dose of 40 mg/kg was followed by a continuous i.p. infusion of 80 micrograms/kg/min and application of 0.4 mM caffeine in the dialysis medium. The results demonstrated that in the rat DG, local administration of 50 mM caffeine significantly stimulates a spontaneous 45Ca2+ efflux and its release induced by 5 mM NMDA. However, systemic administration of caffeine had no effect on spontaneous and NMDA-induced 45Ca2+ release in the rat DG, which supports the view that caffeine, applied in vivo, even in high doses, does not influence CICR in brain neurones.

Protection against post-ischaemic neuronal loss in gerbil hippocampal CA1 by glycineB and AMPA antagonists. Short communication.

Novel antagonists of the glycineB site of the NMDA receptor (MRZ 2/570, MRZ 2/576), and an AMPA receptor antagonist, NBQX were tested in 3-min global ischaemia in gerbils. Untreated animals showed after 14 days a loss of almost 90% of pyramidal neurones in the CA1 region, which was prevented by NBQX, and reduced to 50% by both glycineB antagonists. NBQX produced a delayed, long lasting (up to 24 hr) hypothermia while hypothermia with both glycineB antagonists was transient.

Differential expression of NMDA-evoked 45Ca release in rat hippocampal regions in vivo.

Recently we detected NMDA-induced 45Ca release in the rat dentate gyrus in vivo, attributable to the Ca2+ induced Ca2+ release (CICR) from the endoplasmic reticulum via ryanodine channels. In these experiments we compare expression of NMDA-evoked 45Ca release in the rat dentate gyrus (DG), CA1 and subiculum (SUB). The rationale behind introducing this study is that these hippocampal regions are known to differ in their levels of ryanodine receptors. The release of 45Ca was studied using in vivo microdialysis combined with measurements of 45Ca efflux from prelabelled hippocampal regions. It was shown that NMDA-induced 45Ca release, highly pronounced in the rat DG/CA4, is significantly less expressed in the CA1, whereas in the SUB an NMDA-evoked decrease in 45Ca efflux was noted. This corresponds to distribution of ryanodine receptors in the rat hippocampus, known from the literature. Expression of NMDA-evoked 45Ca release in these rat hippocampal regions which are enriched in ryanodine receptors supports our working hypothesis that CICR via ryanodine channels may be mainly responsible for 45Ca release.

NMDA receptor-mediated calcium fluxes in the hippocampus: relevance to ischemic brain pathology.

Excitotoxic hypothesis of ischemic brain lesion postulates a causal relation between stimulation of glutamatergic receptors and potentially neurotoxic neuronal calcium overload. In this short review we sum up our microdialysis studies on NMDA- and ischemia-evoked CA2+ fluxes in the rabbit hippocampus, which indicate that NMDA channels in normal conditions represent a potential route of Ca2+ influx to the hippocampal neurones, and in forebrain ischemia they significantly contribute to Ca2+ fluxes. These conclusions contrast with other data indicating that NMDA receptors play a marginal role in Ca2+ currents to neurones during advanced forebrain ischemia. A possible inhibition of NMDA receptors in ischemia by acidosis and nitric oxide has been postulated. Our recent data indicate that in brain microdialysis experiments local stabilisation of neutral brain parenchymal pH may prevent acidosis-induced inhibition of NMDA channels during ischemia, leading to overestimation of their role in calcium fluxes.