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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.

The results of genetic studies suggest a possible role for SNAP-25 polymorphism in the development of autism spectrum disorders (ASDs); however, there are no data available on whether changes in SNAP-25 expression also affect animals in rodent models of ASD. The aim of the present study was to explore this issue. The studies included 1-month-old rats representing valproic acid (VPA)- and thalidomide (THAL)-induced models of autism. Their mothers received single doses of VPA (800 mg/kg) or THAL (500 mg/kg) per os on the 11th day of gestation. SNAP-25 protein content in the cerebellum, hippocampus, and frontal lobe was determined using Western blotting, while changes of mRNA levels of Snap25 gene were determined using real-time polymerase chain reaction. Compared to controls, SNAP-25 content was decreased by approximately 35% in all brain structures tested, in both males and females, exclusively in the VPA group. In contrast to this, Snap25 expression, studied in males, was increased in the hippocampus and cerebellum in both, VPA- and THAL-treated rats. We discuss the compliance of these results with the hypothesized role of SNAP-25 in the pathophysiology of ASD and the adequacy of the experimental models used.

Zinc and copper are important trace elements necessary for the proper functioning of neurons. Impaired zinc and/or copper metabolism and signaling are implicated in many brain diseases, including autism (ASD). In our studies, autistic-like behavior in rat offsprings was induced by application to pregnant mothers valproic acid or thalidomide. Zinc and copper contents were measured in serum and brain structures: hippocampus, cerebral cortex, and cerebellum. Our research shows no interconnections in the particular metal concentrations measured in autistic animal brains and their sera. Based on patient researches, we studied 26 genes belonging to disturbed neurotransmitter pathways. In the same brain regions, we examined the expression of genes encoding proteins of cholinergic, adrenergic, serotonin, and dopamine receptors. In both rats’ ASD models, 17 out of the tested gene expression were decreased. In the cerebellum and cerebral cortex, expression of genes encoding cholinergic, adrenergic, and dopaminergic receptors decreased, whereas in the hippocampus only expression of serotoninergic receptors genes was downregulated. The changes in metals content observed in the rat brain can be secondary phenomena, perhaps elements of mechanisms that compensate for neurotransmission dysfunctions.

In the present study, primary cultures of rat cerebellar granule cells (CGC) and the RT2 Profiler PCR array were used to examine the effect of acutely applied brominated flame retardant tetrabromobisphenol A (TBBPA) on the expression of 84 genes related to the main modes of programmed cell death. CGC, at the 7th day of culture, were exposed to 10 or 25μM TBBPA for 30min. Then, 3, 6, and 24h later, the viability of the cells was examined by the staining with propidium iodide (PI) or using the calcein/ethidium homodimer (CA/ET) live/dead kit, and RNA was extracted for the evaluation of gene expression by RT-PCR. At 3, 6 and 24h after the treatment, the number of viable neurons decreased, according to the PI staining method, to 75%, 58% and 41%, respectively, and with the CA/ET method to 65%, 58% and 28%, respectively. In CGC analyzed 3h after the treatment with 25μM TBBPA or 6h after 10μM TBBPA, the only change in the gene expression was a reduction in the expression of Tnf, which is associated with autophagy and may activate some pro-apoptotic proteins. Six hours after 25μM TBBPA, only 2 genes were over-expressed, a pro-apoptotic Tnfrsf10b and Irgm, which is related to autophagy, and the genes that were suppressed included the anti-apoptotic gene Xiap, the necrosis-related Commd4, pro-apoptotic Abl1, 5 genes involved in autophagy (App, Atg3, Mapk8, Pten, and Snca) and 2 genes that participate in two metabolic pathways: Atp6v1g2 (pro-apoptotic and necrosis) and Tnf (pro-apoptotic, autophagy). Autophagy-related Snca and Tnf remained under-expressed 24h after treatment with 25μM TBBPA, which was accompanied by the over-expression of the pro-apoptotic Casp6, the anti-apoptotic Birc3, 2 genes related to autophagy (Htt and Irgm) and 2 genes (Fas and Tp53) that are involved in both apoptosis (pro-apoptotic) and autophagy. These results show a complex pattern of TBBPA-evoked changes in the expression of the genes involved in the programmed neuronal death, indicating no induction of programmed necrosis, an early suppression of the autophagy and anti-apoptotic genes, followed by a delayed activation of genes associated with apoptosis.

The brominated flame retardant tetrabromobisphenol A (TBBPA) has recognized neurotoxic properties mediated by intracellular Ca 2+ imbalance and oxidative stress. Although these factors are known to trigger the release of Zn 2+ from intracellular stores, the effects of TBBPA on Zn 2+ homeostasis in neurons and the role of Zn 2+ in TBBPA neurotoxicity have not yet been studied. Therefore, we investigated zinc transients in primary cultures of rat cerebellar granule cells and assessed their involvement in TBBPA neurotoxicity. The results demonstrate that TBBPA releases Zn 2+ from the intracellular stores and increases its intracellular concentration, followed by Zn 2+ displacement from the cells. TBBPA-evoked Zn 2+ transients are partially mediated by Ca 2+ and ROS. Application of TPEN, Zn 2+ chelator, potentiates TBBPA- and glutamate-induced 45 Ca uptake, enhances TBBPA-induced ROS production and potentiates decreases in the ΔΨm in cells treated with 25 μM TBBPA, revealing the potential neuroprotective capacity of endogenous Zn 2+ . However, the administration of TPEN does not aggravate TBBPA neurotoxicity, and even slightly decreases neuronal death induced by 25 μM TBBPA. In summary, it was shown for the first time that TBBPA interferes with the cellular Zn 2+ homeostasis in neuronal cultures, and we revealed complex roles for endogenous Zn 2+ in cytoprotection and TBBPA toxicity in cultured neurons.

Autism spectrum disorders (ASD) include neurodevelopmental disorders in which behavioral deficits can result from neuronal imbalance of excitation to inhibition (E/I) in the brain. Here we used RT-qPCR to screen for the expression of 99 genes associated with excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmission in the cerebral cortex, hippocampus and cerebellum of rats in an established VPA model of ASD. The largest changes in the expression of glutamatergic genes were found in the cerebral cortex, where 12 genes including these encoding some of the subunits of the ionotropic glutamate receptors, were upregulated, while 2 genes were downregulated. The expression of genes encoding the presynaptic glutamatergic proteins vGluT1 and mGluR7 and PKA, involved in downstream glutamatergic signaling, was elevated more than 100-fold. Changes in GABAergic gene expression were found in the cortex, cerebellum and hippocampus; 3 genes were upregulated, and 3 were downregulated. In conclusion, these results revealed that, in the ASD model, several glutamatergic genes in the rat cerebral cortex were upregulated, which contrasts with small and balanced changes in the expression of GABAergic genes. The VPA rat model, useful in studying the molecular basis of ASD, may be suitable for testing experimental therapies in these disabilities.

The brominated flame retardant tetrabromobisphenol A (TBBPA) is toxic to cultured brain neurons, and glutamate receptors partially mediate this effect; consequently, the depolarizing effect of TBBPA on neurons is to be expected, but it is yet to be actually demonstrated. The aim of this study was to detect TBBPA-evoked depolarization and identify the underlying mechanisms. The plasma membrane potential of rat cerebellar granule cells (CGC) in cerebellar slices or in primary cultures was measured using whole-cell current clamp recordings, or the fluorescent probe oxonol VI, respectively. The contribution of NMDA and AMPA receptors, voltage-gated sodium channels and intracellular calcium mobilization was tested using their selective antagonists or inhibitors. Direct interactions of TBBPA with NMDARs were tested by measuring the specific binding of radiolabeled NMDAR ligands to isolated rat cortical membrane fraction. TBBPA (25 μM) strongly depolarized CGC in cerebellar slices, and at ≥ 7.5 μM concentration-dependently depolarized primary CGC cultures. Depolarization of the primary CGC by 25 μM TBBPA was partly reduced when MK-801 was applied alone or in combination with either TTX or CNQX, or where bastadin 12 was applied in combination with ryanodine, whereas depolarization was completely prevented when MK-801, CNQX and TTX where combined. TBBPA had no effect on the specific binding of NMDAR radio-ligands to isolated cortical membranes. These results demonstrate the depolarizing effect of TBBPA on CGC, which is mainly mediated by ionotropic glutamate receptors, while voltage-gated sodium channels are also involved. We found no evidence for the direct activation of NMDARs by TBBPA.

The disorders of the glutamatergic neurotransmission have been associated with pathogenesis of autism. In this study we evaluated the impact of the in vivo and ex vivo test methodology on measurements of levels of neurotransmitter amino acids in hippocampus of rats for valproic acid- (VPA) and thalidomide- (THAL) induced models of autism. The main goal was to compare the changes in concentrations of glutamate (Glu), glutamine (Gln) and GABA between both autistic groups and the control, measured in vivo and ex vivo in homogenates. The rat pups underwent three in vivo tests: ultrasonic vocalization (USV), magnetic resonance spectroscopy (MRS) and unilateral microdialysis of the hippocampus. Analyses of homogenates of rat hippocampus were performed using high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. For the statistical analysis, we performed univariate and multivariate tests. USV test, which is considered in rodents as an indicator of pathology similar to autism, showed decreased USV in VPA and THAL groups. In vivo MRS studies demonstrated increases of Glu content in male rat’s hippocampus in VPA and THAL groups, while the microdialysis, which allows examination of the contents in the extracellular space, detected decreases in the basal level of Gln concentrations in VPA and THAL groups. Ex vivo HPLC studies showed that levels of Glu, Gln and GABA significantly increased in male rat’s hippocampus in the VPA and THAL groups, while NMR studies showed increased levels of Gln and GABA in the VPA group. Collectively, these results are consistent with the hypothesis suggesting the role of the glutamatergic disturbances on the pathogenesis of autism. For all methods used, the values of measured changes were in the same direction. The orthogonal partial least square discriminant analysis confirmed that both animal models of autism tested here can be used to trace neurochemical changes in the brain.

The study assessed involvement of Ca(2+) signaling mediated by the metabotropic glutamate receptors mGluR1/5 in brain tolerance induced by hypoxic preconditioning. Acute slices of rat piriform cortex were tested 1 day after exposure of adult rats to mild hypobaric hypoxia for 2 h at a pressure of 480 hPa once a day for three consecutive days. We detected 44.1 ± 11.6 % suppression of in vitro anoxia-induced increases of intracellular Ca(2+) levels and a fivefold increase in Ca(2+) transients evoked by selective mGluR1/5 agonist, DHPG. Western blot analysis of cortical homogenates demonstrated a 11 ± 4 % decrease in mGluR1 immunoreactivity (IR), and in the nuclei-enriched fraction a 12 ± 3 % increase in IR of phospholipase Cβ1 (PLCβ1), which is a major mediator of mGluR1/5 signaling. Immunocytochemical analysis of the cortex revealed increase in the mGluR1/5 and PLCβ1 IR in perikarya, and a decrease in IR of the neuronal inositol trisphosphate receptors (IP3Rs). We suggest that enhanced expression of mGluR5 and PLCβ1 and potentiation of Ca(2+) signaling may represent pro-survival upregulation of Ca(2+)-dependent genomic processes, while decrease in mGluR1 and IP3R IR may be attributed to a feedback mechanism preventing excessive intracellular Ca(2+) release.

The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca(2+) transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, (45)Ca uptake was measured, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of (45)Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca(2+) and the NMDAR-mediated influx of Ca(2+) into neurons participate in the mechanism of TBBPA-induced Ca(2+) imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.

Introduction: In vitro experiments have demonstrated that preconditioning primary neuronal cultures by temporary application of NMDA receptor antagonists induces long-term tolerance against lethal insults. In the present study we tested whether similar effects also occur in brain submitted to ischemia in vivo and whether the potential benefit outweighs the danger of enhancing the constitutive apoptosis in the developing brain. Material and methods: Memantine in pharmacologically relevant doses of 5 mg/kg or (+)MK-801 (3 mg/kg) was administered i.p. 24, 48, 72 and 96 h before 3-min global forebrain ischemia in adult Mongolian gerbils or prior to hypoxia/ ischemia in 7-day-old rats. Neuronal loss in the hippocampal CA1 in gerbils or weight deficit of the ischemic hemispheres in the rat pups was evaluated after 14 days. Also, the number of apoptotic neurons in the immature rat brain was evaluated. Results: In gerbils only the application of (+)MK-801 24 h before ischemia resulted in significant prevention of the loss of pyramidal neurons. In rat pups administration of (+)MK-801 at all studied times before hypoxia-ischemia, or pretreatment with memantine or with hypoxia taken as a positive control 48 to 92 h before the insult, significantly reduced brain damage. Both NMDA receptor antagonists equally reduced the number of apoptotic neurons after hypoxia-ischemia, while (+)MK-801-evoked potentiation of constitutive apoptosis greatly exceeded the effect of memantine. Conclusions: We ascribe neuroprotection induced in the immature rats by the pretreatment with both NMDA receptor antagonists 48 to 92 h before hypoxia-ischemia to tolerance evoked by preconditioning, while the neuroprotective effect of (+)MK-801 applied 24 h before the insults may be attributed to direct consequences of the inhibition of NMDA receptors. This is the first report demonstrating the phenomenon of inducing tolerance against hypoxia-ischemia in vivo in developing rat brain by preconditioning with NMDA receptor antagonists.

A presynaptic protein SNAP-25 belonging to SNARE complex which is instrumental in intracellular vesicular trafficking and exocytosis, has been implicated in hyperactivity and cognitive abilities in some neuropsychiatric disorders. The unclear etiology of the behavior disrupting neurodevelopmental disabilities in addition to genetic causes most likely involves environmental factors. The aim of this in vitro study was to test if various suspected developmental neurotoxins can alter SNAP-25 mRNA and protein expression in neurons. Real-time PCR and Western blotting analyses were used to assess SNAP-25 mRNA and protein levels in primary cultures of rat cerebellar granule cells (CGCs). The test substances: tetrabromobisphenol-A (TBBPA), thimerosal (TH), silver nanoparticles (NAg), valproic acid (VPA) and thalidomide (THAL), were administered to CGC cultures at subtoxic concentrations for 24 h. The results demonstrated that SNAP-25 mRNA levels were increased by 49 and 66% by TBBPA and THAL, respectively, whereas VPA and NAg reduced these levels to 48 and 64% of the control, respectively. The SNAP-25 protein content in CGCs was increased by 79% by TBBPA, 25% by THAL and 21% by NAg; VPA and TH reduced these levels to 73 and 69% of the control, respectively. The variety of changes in SNAP-25 expression on mRNA and protein level suggests the diversity of the mechanism of action of the test substances. This initial study provided no data on concentration-effect relations and on functional changes in CGCs. However it is the first to demonstrate the effect of different compounds that are suspected of causing neurodevelopmental disabilities on SNAP-25 expression. These results suggest that this protein may be a common target for not only inherited but also environmental modifications linked to behavioral deficits in neurodevelopmental disabilities.

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca2+]i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (∆Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca2+]i and ROS production, and reduced GSH content, catalase activity, ∆Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca2+ and drop in ∆Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca2+-independent portion of oxidative stress and cytotoxicity emerges. Electronic supplementary material The online version of this article (doi:10.1007/s11064-016-2075-x) contains supplementary material, which is available to authorized users.

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer’s disease (AD) including memory deficit, presence of β-amyloid (Aβ) oligomers, and age-associated formation of amyloid deposits. In this study we used hippocampal microdialysis to characterize the signaling of N-methyl- d -aspartic acid receptors (NMDA-Rs) in awake and behaving AD Tg mice. The NMDA-R signaling is central to hippocampal synaptic plasticity underlying memory formation and several lines of evidence implicate the role of Aβ oligomers in effecting NMDA-R dysfunction. CA1 NMDA-Rs were stimulated by NMDA infused through reverse microdialysis while changes in the cyclic guanosine monophosphate (cGMP) concentration in the brain interstitial fluid (ISF) were used to determine NMDA-Rs responsiveness. While 4 months old wild type C57BL/6 mice mounted robust cGMP response to the NMDA challenge, the same stimulus failed to significantly change the cGMP level in 4 and 15 months old APPSW and 4 months old APPSW/PS1L166P Tg mice, which were all on C57BL/6 background. Lack of response to NMDA in AD Tg mice occurred in the absence of changes in expression levels of several synaptic proteins including synaptophysin, NR1 NMDA-R subunit and postsynaptic density protein 95, which indicates lack of profound synaptic degeneration. Aβ oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5 ± 3.6 ng/g in 4 months old APPSW mice to 60.8 ± 15.9 ng/g in 4 months old APPSW/PS1L166P mice. Four months old APPSW mice had no Aβ amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient Aβ amyloid plaque formation. Our studies describes a novel approach useful to study the function of NMDA-Rs in awake and behaving AD Tg mice and demonstrate impairment of NMDA-R response in the presence of endogenously formed Aβ oligomers but predating onset of Aβ amyloidosis.

Preconditioning with sublethal ischemia or hypoxia may reduce the high susceptibility of CA1 pyramidal neurons to ischemic injury. In this study, we tested the hypothesis that enhanced level of neuropeptide Y (NPY) might play a role in the mechanisms responsible for this induced tolerance. Changes in NPY immunoreactivity in the hippocampal formation of preconditioned Mongolian gerbils were compared with the level of tolerance to test ischemia. Tolerance was induced by preconditioning with 2-min of ischemia or with three trials of mild hypobaric hypoxia (360 Torr, 2 h), separated by 24 h, that were completed 48 h before the 3-min test ischemia. The number of NPY-positive neurons in the gerbil hippocampal formation was assessed 2, 4 and 7 days after preconditioning. Survival of the CA1 pyramidal neurons was examined 14 days after the insult. Our experiments demonstrated that ischemic and hypoxic preconditioning produced equal attenuation of the damage evoked by 3-min ischemia, although the pattern of NPY immunoreactivity in the hippocampus differed. Preconditioning ischemia resulted in a 20% rise in the number of NPY-positive neurons 2 days later that disappeared 4 days after the ischemic episode, while mild hypobaric hypoxia induced a twofold increase in the number of NPY-positive neurons that lasted for at least 7 days. Although induced tolerance to ischemia 2 days after ischemic or hypoxic preconditioning was accompanied by increased immunoreactivity of NPY, there was no correlation between its intensity and the level of neuroprotection.

In this study we tested the hypothesis that dantrolene, an established inhibitor of the skeletal muscle isoform of the ryanodine receptor, may interfere with activity of NMDA receptors in neurons. We assessed the effects of dantrolene on [(3)H]MK-801 and [(3)H]glycine binding to isolated rat cortical membranes. Dantrolene inhibited [(3)H]MK-801 binding in the presence of 100 microM NMDA with an IC(50) of 58.4 microM. The IC(50) value increased to 99.6, 343.0 and 364.6 microM in the presence of 10, 30 and 50 microM glycine, respectively, suggesting that dantrolene competes with glycine for binding site at the NMDA receptor complex. A binding assay using [(3)H]glycine confirmed this supposition: dantrolene inhibited strychnine-insensitive glycine binding in a dose-dependent way. Thus, our results show that dantrolene at concentrations of 50-100 microM and higher blocks the glycine binding site of the NMDA receptor complex and in this way inhibits activation of the NMDA ion channel. These data reveal a new mechanism of dantrolene action in neuronal tissue. Our results also suggest that the neuroprotective effect of dantrolene may be at least partly explained by its activity as a non-competitive antagonist of NMDA receptors.

In this study we tested the effect of antagonists of two subtypes of the group I metabotropic glutamate receptors (mGluRs GI) on the induction of ischemic tolerance in relation to brain temperature. These experiments were prompted by indications that glutamate receptors may participate in the mechanisms of ischemic preconditioning. The role of NMDA receptors in the induction of ischemic tolerance has been debated while there is lack of information concerning the involvement of mGluRs GI in this phenomenon. The tolerance to injurious 3 min forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Brain temperature was measured using telemetry equipment. EMQMCM and MTEP, antagonists of mGluR1 and mGluR5, respectively, were injected i.p. at a dose of 5 mg/kg. They were administered either before preconditioning ischemia in a single dose or after 2 min ischemia three times every 2 h. Both antagonists did not inhibit the induction of ischemic tolerance. Thus, our data indicate that group I metabotropic glutamate receptors do not play an essential role in the induction of ischemic tolerance.

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4-tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free-radical generation in an abiotic system, as well as indices of neurotoxicity (caspase-3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7-day-old rats, 1MeTIQ prevented the glutamate-induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK-801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate-induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.

Increased serum level of homocysteine, a sulfur-containing amino acid, is considered a risk factor in vascular disorders and in dementias. The effect of homocysteine and metabolically related compounds on brain production of kynurenic acid (KYNA), an endogenous antagonist of glutamate ionotropic receptors, was studied. In rat cortical slices, DL-homocysteine enhanced (0.1-0.5 mM) or inhibited (concentration inducing 50% inhibition [IC50]=6.4 [5.5-7.5] mM) KYNA production. In vivo peripheral application of DL-homocysteine (1.3 mmol/kg intraperitoneally) increased KYNA content (pmol/g tissue) from 8.47 +/- 1.57 to 13.04 +/- 2.86 (P0.01; 15 min) and 11.4 +/- 1.72 (P0.01; 60 min) in cortex, and from 4.11 +/- 1.54 to 10.02 +/- 3.08 (P0.01; 15 min) in rat hippocampus. High concentrations of DL-homocysteine (20 mM) applied via microdialysis probe decreased KYNA levels in rabbit hippocampus; this effect was antagonized partially by an antagonist of group I metabotropic glutamate receptors, LY367385. In vitro, S-adenosylhomocysteine acted similar to but more potently than DL-homocysteine, augmenting KYNA production at 0.03-0.08 mM and reducing it ator =0.5 mM. The stimulatory effect of S-adenosylhomocysteine was abolished in the presence of the L-kynurenine uptake inhibitors L-leucine and L-phenyloalanine. Neither the N-methyl-D-aspartate (NMDA) antagonist CGS 19755 nor L-glycine influenced DL-homocysteine- and S-adenosylhomocysteine-induced changes of KYNA synthesis in vitro. DL-Homocysteine inhibited the activity of both KYNA biosynthetic enzymes, kynurenine aminotransferases (KATs) I and II, whereas S-adenosylhomocysteine reduced only the activity of KAT II. L-Methionine and L-cysteine, thiol-containing compounds metabolically related to homocysteine, acted only as weak inhibitors, reducing KYNA production in vitro and inhibiting the activity of KAT II (L-cysteine) or KAT I (L-methionine). The present data suggest that DL-homocysteine biphasically modulates KYNA synthesis. This seems to result from conversion of compound to S-adenosylhomocysteine, also acting dually on KYNA formation, and in part from the direct interaction of homocysteine with metabotropic glutamate receptors and KYNA biosynthetic enzymes. It seems probable that hyperhomocystemia-associated brain dysfunction is mediated partially by changes in brain KYNA level.

Calbindin D 28K (CB) expression was analyzed in the rat hippocampus following 10-min-cardiac arrest-induced ischemia within a year after reperfusion. In rats examined 3 days after ischemia, CB immunoreactivity disappeared completely from CA1 pyramidal neurons and from most CA2 pyramids. In the stratum granulosum of the dentate gyrus, mossy fibers, and hippocampal interneurons, CB immunoreactivity was preserved, although staining was somewhat paler than that in control rats. A similar pattern of CB immunoreactivity was found in rats sacrificed 14 days and 1 month after cardiac arrest. From the 14 th postischemic day, neuronal loss in the stratum pyramidale of CA1 but not in that of CA2 became apparent. The reappearance of CB immunoreactivity in CA1 and CA2 pyramidal neurons was noticed 6 months after ischemia, and the pattern was identical to that observed in animals sacrificed 12 months after the ictus. The prolonged loss and delayed reappearance of CB immunoreactivity in the hippocampus demonstrate that ischemia may induce long-term disturbances of protein expression, which may in turn result in impairment of hippocampal functioning.

In vitro experiments have demonstrated that preconditioning primary neuronal cultures by temporary application of NMDA receptor antagonists induces long-term tolerance against lethal insults. In the present study we tested whether similar effects also occur in brain submitted to ischemia in vivo and whether the potential benefit outweighs the danger of enhancing the constitutive apoptosis in the developing brain.Memantine in pharmacologically relevant doses of 5 mg/kg or (+)MK-801 (3 mg/kg) was administered i.p. 24, 48, 72 and 96 h before 3-min global forebrain ischemia in adult Mongolian gerbils or prior to hypoxia/ischemia in 7-day-old rats. Neuronal loss in the hippocampal CA1 in gerbils or weight deficit of the ischemic hemispheres in the rat pups was evaluated after 14 days. Also, the number of apoptotic neurons in the immature rat brain was evaluated.In gerbils only the application of (+)MK-801 24 h before ischemia resulted in significant prevention of the loss of pyramidal neurons. In rat pups administration of (+)MK-801 at all studied times before hypoxia-ischemia, or pretreatment with memantine or with hypoxia taken as a positive control 48 to 92 h before the insult, significantly reduced brain damage. Both NMDA receptor antagonists equally reduced the number of apoptotic neurons after hypoxia-ischemia, while (+)MK-801-evoked potentiation of constitutive apoptosis greatly exceeded the effect of memantine.We ascribe neuroprotection induced in the immature rats by the pretreatment with both NMDA receptor antagonists 48 to 92 h before hypoxia-ischemia to tolerance evoked by preconditioning, while the neuroprotective effect of (+)MK-801 applied 24 h before the insults may be attributed to direct consequences of the inhibition of NMDA receptors. This is the first report demonstrating the phenomenon of inducing tolerance against hypoxia-ischemia in vivo in developing rat brain by preconditioning with NMDA receptor antagonists.

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.

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 45 Ca from the pre-labeled endogenous Ca 2+ pool was observed and interpreted as the expression of intracellular Ca 2+ release. Dantrolene (100 μM) significantly inhibited the NMDA-induced 45 Ca release, whereas 250 μM 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 [ 3 H]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 45 Ca 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 45 Ca 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 45 Ca release is negligible.

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

et al., Treatment of patients with acute liver failure (ALF) is unsatisfactory and mortality remains unacceptably high. Blocking NMDA receptors delays or prevents death of rats with ALF. The underlying mechanisms remain unclear. Clarifying these mechanisms will help to design more efficient treatments to increase patient's survival. The aim of this work was to shed light on the mechanisms by which blocking NMDA receptors delays rat's death in ALF. ALF was induced by galactosamine injection. NMDA receptors were blocked by continuous MK-801 administration. Edema and cerebral blood flow were assessed by magnetic resonance. The time course of ammonia levels in brain, muscle, blood, and urine; of glutamine, lactate, and water content in brain; of glomerular filtration rate and kidney damage; and of hepatic encephalopathy (HE) and intracranial pressure was assessed. ALF reduces kidney glomerular filtration rate (GFR) as reflected by reduced inulin clearance. GFR reduction is due to both reduced renal perfusion and kidney tubular damage as reflected by increased Kim-1 in urine and histological analysis. Blocking NMDA receptors delays kidney damage, allowing transient increased GFR and ammonia elimination which delays hyperammonemia and associated changes in brain. Blocking NMDA receptors does not prevent cerebral edema or blood-brain barrier permeability but reduces or prevents changes in cerebral blood flow and brain lactate. The data show that dual protective effects of MK-801 in kidney and brain delay cerebral alterations, HE, intracranial pressure increase and death. NMDA receptors antagonists may increase survival of patients with ALF by providing additional time for liver transplantation or regeneration. © 2013 Springer Science+Business Media New York., Supported by grants from Ministerio de Ciencia Innovacion Spain (SAF2008-00062, SAF2011-23051; CSD2008-00005; PS09/00806; PI10/01434) and Consellería Educación (PROMETEO-2009-027; ACOMP/2011/053; ACOMP/2012/066) and Conselleria Sanitat (AP-043-10, AP-004/11, AP-087/11) Generalitat Valenciana.

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.

Alzheimer’s disease (AD) is associated with increased brain levels of β-amyloid (Aβ) peptides, which readily self-aggregate into fibrils and oligomers that have particularly deleterious properties towards synapses of excitatory glutamatergic neurons. Here, we examined the neuroprotective effects of 1-methyl-1,2,3,4,-tetrahydroisoquinoline (1MeTIQ) against Aβ-induced loss of synaptic proteins in cultured primary hippocampal neurons. Exposure of mature primary hippocampal neurons to 10 μM synthetic Aβ1-40 over 72 h resulted in approximately 60% reduction in the surface expression of NR1 subunit of the NMDA receptor (NMDAR), PSD-95, and synaptophysin, without causing neuronal death. Concomitant treatment with 500 μM of 1MeTIQ, a low affinity NMDAR antagonist significantly ameliorated the loss of synaptic protein markers. The neuroprotective properties of 1MeTIQ were compared with those of MK-801, which at 0.5 μM concentration also prevented Aβ1-40-induced loss of synaptic proteins in primary neuronal cultures. Furthermore, we provide novel evidence demonstrating effectiveness of 1MeTIQ in reducing the level of reactive oxygen species (ROS) in primary neuronal culture system. As oxidative stress contributes importantly to neurodegeneration in AD, 1MeTIQ may provide a dual neuroproctective effect in AD both as a NMDARs antagonist and ROS formation inhibitor. 1MeTIQ occurs endogenously at low concentrations in the brain and its synthetic form readily penetrates the blood-brain-barrier after the systemic administration. Our results highlight a possibility of the application of 1MeTIQ as a neuroprotective agent in AD related neurodegeneration.

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 1H 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.

A photochemical ring model of ischaemia was introduced in the middle of the nineteen eighties. Irradiation by a laser or arc lamp followed by intravenous injection of rose bengal resulted in thrombosis of pial and superficial cortical vessels. This ring model imitated focal ischaemic damage in humans. In our experiment twenty-seven Wistar rats of both sexes weighing 250-300 grams were examined. A photochemical ring model based on irradiation of the area of parietal bone 4 mm posteriorly to the bregma and 4 mm laterally from the sagittal suture was applied. A ring-shaped light beam with a wavelength of 510-540 nm with 5 mm diameter was generated by a high pressure discharge lamp at a power of 400 W. Two groups of rats treated and untreated with MK-801 and two rings of the thickness of 0.35 mm and 0.5 mm were used in the experiment. Morphological examination was performed in animals sacrificed 1 and 4 days after the irradiation. On formalin-fixed and paraffin-embedded slices HE staining method and immunoreaction with antibodies to ubiquitin were applied. Our material confirmed well known information about the dynamics of infarct breakdown, ischaemic-induced angiogenesis, glial reaction and other typical changes described previously in handbooks and numerous papers. In the experiment, morphological changes were more intensive after the irradiation by 0.5 mm than 0.35 mm irradiating rings and 4 days than one day after the irradiation. A surprising finding observed in some of the examined animals was more intensive neuronal damage after treatment with MK-801. Another unpredicted discovery was intensive morphological alterations found in CA4 and CA3 hippocampal sectors. Moreover, these alterations were not limited to the damaged hemisphere, but were also observed contralaterally. In some of the rats, ischaemic and necrotic cells were additionally found within both parasagittal areas. We connect this atypical localization of the ischaemic changes with dispersion of light emitted by the used lamp. Dispersed light also leads to thrombotic occlusion of the meningeal arteries in the parasagittal area. Among these arteries, thrombosis in pericallosal and penetrating arteries was present. Our experiment demonstrated that if a non-laser lamp is used, brain areas distant from the necrotic ring must be carefully investigated.

Our results demonstrate that acute exposure of primary rat cerebellar granule cell cultures to homocysteine at millimolar concentrations induces a glutamate receptor-mediated decrease in tau protein phosphorylation, which is accompanied by excitotoxic neuronal damage. This contrasts with the previously reported hyperphosphorylation of tau in homocysteine-treated neurons, and with the assumption that hyperhomocysteinemia is one of the risk factors in Alzheimer disease, in which abnormal hyperphosphorylation of tau protein leads to neurofibrillary degeneration. The mechanisms of homocysteine neurotoxicity have been explained mainly either by disturbances in methylation processes, that may also lead to the accumulation of phosphorylated tau due to reduced activity of tau-selective protein phosphatase 2A, or by excitotoxicity. Since the relationships between homocysteine excitotoxicity and tau phosphorylation are unclear, the aim of this study was to characterize these processes in neurons acutely treated with homocysteine at neurotoxic concentrations, and to link them to the activities of glutamate receptors and protein phosphatase 2A. Within 24h following a 30 min exposure of neuronal cultures to 20mM d,l-homocysteine, significant neurotoxicity was induced. This could be reduced by treatment with an uncompetitive NMDA receptor antagonist, MK-801 (0.5 microM), or by mGlu1 and mGlu5 receptor antagonists, LY367385 and MPEP, respectively (both at 25 microM). Western blot analysis showed a rapid decrease in immunostaining of phospho-tau, 2h after incubation of cell cultures with 15 mM D,L-homocysteine, which persisted for 6h after the insult. Application of MK-801, LY367385 or okadaic acid (100 nM), an inhibitor of protein phosphatases 1 and 2A, significantly prevented dephosphorylation of tau, implying a role for the activation of glutamate receptors and protein phosphatase 2A. The phosphorylation of tau may be increased or reduced by treatment with homocysteine, and the nature of the cellular response to this sulfur-containing amino acid depends on the neuronal phenotype.

Constraining the catechol aryl ether moiety of bastadins by incorporation into a macrocyle is not necessary in order to mimic the effects of these marine natural products on neuronal calcium homeostasis. Simple, acyclic analogs that embody the 'western' or 'eastern' parts of bastadins were found to evoke comparable responses with bastadin 5.

The aims of this study were (1) to characterize calcium signaling in rat cortex induced by repeated in vitro application of the glutamatergic agonists L-glutamate, NMDA, AMPA and DHPG, (2) to analyze the influence of transient severe hypobaric hypoxia (180 Torr) administered in vivo on calcium responses to stimulation of glutamate receptors by their agonists, and (3) to evaluate the effects of preconditioning with intermittent mild hypobaric hypoxia (360 Torr) 24 h before the severe hypoxia, on these Ca2+ responses. Intracellular Ca2+ dynamics was studied using the fluorescent probes fura-2 and chlortetracycline to monitor free and bound calcium (Cai and Cab) respectively. In control cortical slices, application of L-glutamate, NMDA and AMPA induced concomitant increases in Cai and Cab, reflecting Ca2+ influx and its intracellular accumulation in neurons. DHPG, an agonist of group I mGlu receptors induced a decrease in Cab accompanied by a rise in Cai levels, indicating Ca2+ mobilization. In cortical slices collected 24 h after severe hypoxia, the responses of Cab to glutamate administration were increased, DHPG-induced shifts were reversed, the increase in Cab after the first application of AMPA was reduced, while after the second, Cab rises were potentiated, and the increases in Cab evoked by NMDA application were slightly suppressed. The alterations of responses in Cab to the selective agonists were completely prevented by preconditioning with mild hypoxia. Our results suggest that protection of normal glutamatergic calcium signaling contributes to tolerance to hypoxia induced by preconditioning.

Nicotinamide (NAM), an important cofactor in many metabolic pathways, exhibits at high doses neuroprotective abilities of an unclear mechanism. In the present study we evaluated the unknown protective capability of its immediate metabolite 1-methylnicotinamide (MNA) in comparison to NAM in primary cultures of rat cerebellar granule cells (CGC) submitted to acute excitotoxicity. Neurotoxicity was evaluated with propidium iodide staining 24 h after 30 min exposure to glutamate (GLU) and NMDA. NAM and MNA reduced NMDA toxicity only at 25 mM concentration, while neurotoxicity of 0.5 mM GLU was slightly diminished only by 25 mM NAM. Both compounds at 25 mM reduced GLU-induced 45Ca uptake and dose-dependently inhibited NMDA-induced 45Ca accumulation. Neither NAM nor MNA interfered with GLU-evoked intracellular calcium transients evaluated with calcium orange fluorescent probe or inhibited [3H]MK-801 binding to rat cortical membranes. NAM and MNA failed to change GLU-evoked decrease in mitochondrial membrane potential monitored using the fluorescent dye rhodamine 123. Analysis with a hydroperoxide-sensitive fluorescent probe demonstrated significant reduction by 20 and 25 mM MNA, but not NAM, of oxidative stress in cultures after 1 h treatment with GLU. CGC accumulated radiolabelled NAM and MNA in a time and concentration dependent manner, NAM being transported more rapidly. These findings demonstrate that weak neuroprotective ability of MNA in excitotoxicity, accompanied by incomplete stabilization of calcium imbalance and lessening of oxidative stress, is not connected with direct inhibition of NMDA receptors. The exact mechanisms of these effects require further investigation.

Although the interactions of several natural bastadins with the RyR1 isoform of the ryanodine receptor in sarcoplasmic reticulum has been described, their structure-dependent interference with the RyR2 isoform, mainly expressed in cardiac muscle and brain neurons, has not been studied. In this work, we examined calcium transients induced by natural bastadin 10 and several synthetic bastadins in cultured cerebellar granule cells known to contain RyR2. The fluorescent calcium indicator fluo-3 and confocal microscopy were used to evaluate changes in the intracellular Ca(2+) concentration (Ca(i)), and the involvement of ryanodine receptors was assessed using pharmacological tools. Our results demonstrate that apart from the inactive BAST218F6 (a bisdebromo analogue of bastadin 10), synthetic bastadin 5, and synthetic analogues BAST217B, BAST240 and BAST268 (at concentrations20 microM) increased Ca(i) in a concentration-dependent, ryanodine- and FK-506-sensitive way, with a potency significantly exceeding that of 20 mM caffeine. Moreover, the same active bastadins at a concentration of 5 muM in the presence of ryanodine prevented a thapsigargin-induced increase in Ca(i). These results indicate that bastadins, acting in a structure-dependent manner, modify the activity of RyR2 in primary neuronal culture and provide new information about structure-related pharmacological properties of bastadins.

Elevated homocysteine is a risk factor in cardiovascular diseases and neurodegeneration. Among the putative mechanisms of homocysteine-evoked neurotoxicity, disturbances in methylation processes and NMDA receptor-mediated excitotoxicity have been suggested. Our previous studies demonstrated that group I metabotropic glutamate receptors along with NMDA receptors participate in acute homocysteine-induced neuronal damage. In this study, using propidium iodide staining, we tested whether the same mechanism may mediate chronic homocysteine neurotoxicity. Our results confirmed that the application of D,L-homocysteine in micromolar concentrations for 3 days induces neurodegeneration in primary cultures of cerebellar granule neurons. Uncompetitive NMDA receptor antagonist MK-801, and mGlul or mGlu5 receptor antagonists (LY367385 and MPEP, respectively), given alone provided very limited neuroprotection. However, simultaneous application of the NMDA receptor antagonists MK-801, memantine or amantadine and MPEP almost completely prevented chronic homocysteine neurotoxicity. These findings suggest a novel therapeutic strategy to combat neurodegeneration induced by hyperhomocysteinemia comprising a combination of antagonists of group 1 metabotropic glutamate receptors and NMDA receptors.

In Mongolian gerbils, global forebrain ischemia induces enhanced locomotor activity and the disruption of nest building immediately after the insult, followed by damage to hippocampal neurons developing 3 days later. Preconditioning by a brief episode of sublethal ischemia induces the protection of CA1 hippocampal neurons against a lethal ischemic insult. We examined how preconditioning with 2-min ischemia affects disturbances in the nest building behavior and locomotor activity induced by the injurious 3-min ischemia. Morphological examination confirmed that preconditioning significantly reduced neuronal damage in CA1 evoked by injurious ischemia. Behavioral studies demonstrated that preconditioning reduced the locomotor hyperactivity and latency in nest building after test ischemia, in comparison to sham or naive animals. The results indicate that the nest building test and measurement of locomotor activity may be used for an early in vivo prediction of the extent of ischemic brain damage and tolerance induced by ischemic preconditioning.

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4-tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free-radical generation in an abiotic system, as well as indices of neurotoxicity (caspase-3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7-day-old rats, 1MeTIQ prevented the glutamate-induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK-801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate-induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.

In this study we tested if calcium imbalance and mitochondrial dysfunction, which have been implicated in the conventional mechanisms of excitotoxicity induced by glutamate (Glu), are also involved in homocysteine (Hcy) neurotoxicity. Primary cultures of rat cerebellar granule cells were incubated for 30 min in the presence of 25 mM d , l -Hcy or 1 mM Glu. At these concentrations both amino acids induced comparable neurodegeneration and chromatin condensation, evaluated after 24 h using the propidium iodide and Hoechst 33258 staining. These effects were partially prevented by cyclosporin A (CsA), but not FK506. Hcy-induced release of [ 3 H]inositol phosphates and increase in intracellular calcium level (evaluated with fluo-3 fluorescent probe) were weakly expressed. Hcy- and Glu-induced mitochondrial swelling was visualized under electron microscope, and the release of Cytochrome c was evaluated using immunocytochemical method and confocal microscopy. Comparing to Glu, the effects of Hcy were slightly less expressed and less sensitive to CsA, while FK506 did not modify mitochondrial alterations. These data indicate that mitochondrial alterations play a similar role in acute Hcy and Glu neurotoxicity, although the mechanisms triggering Glu- and Hcy-evoked mitochondrial dysfunction seem to differ, Hcy toxicity being less dependent on calcium.

Summary The neuroprotective efficacy of 2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid-I (ABHxD-I), a rigid agonist of metabotropic glutamate receptors, was studied using a 3-min global cerebral ischaemia model in Mongolian gerbils and the hypoxia/ischaemia model in 7-day-old rats. The effects on brain damage of ABHxD-I (30 mg/kg, intraperitoneally or 7.5 μg intracerebroventricularly) administered 30 min before global ischaemia or 30 min after hypoxia/ischaemia was evaluated 14 days after the insults. Treatment of adult gerbils with ABHxD-I injected i.c.v. but not systemically, prevented post-ischaemic hyperthermia and substantially reduced brain damage. These effects may reflect low permeability of the adult blood–brain barrier to ABHxD-I, and the role of reduced body and brain temperature in neuroprotection after its i.c.v. administration. ABHxD-I given either i.p. or i.c.v. to developing rats reduced brain damage by 55 and 37%, respectively, without affecting the body temperature. Due to immaturity and increased post-ischaemic permeability of the blood–brain barrier in developing rats, ABHxD-I may induce neuroprotection by direct interference with brain metabotropic glutamate receptors.

The mechanism of cytotoxic effects of hyperglycaemia on the brain has not yet been explained and the proposed hypotheses are not fully convincing. In the present study, we aimed to assess the effect of high doses of glucose on the ultrastructure of the mice brain. The results, which are in agreement with the literature data, show that the administration of a single high dose of glucose, as well as its chronic application, leads to accumulation of glycogen granules in the cytoplasm of astrocytes. A new observation is the detection of glycogen granules in the ultrastructurally changed mitochondria of astrocytes as well as in the mitochondria of some synapses. Our hypothesis assumes that excess of glucose may cause an increase in the vulnerability of the brain mitochondria. This in turn may enable glucose and cytoplasmic enzymes to penetrate into the mitochondria and they therein synthesise glycogen. Mitochondrial dysfunction may in turn lead to neurodegeneration by apoptotic process.

This in vivo microdialysis study compared the effects of NMDA and d,l-homocysteine (Hcy) administered via dialysis medium on 45Ca efflux from prelabeled rabbit hippocampus. Application of these agonists evoked dose-dependent, and sensitive to MK-801, opposite effects: NMDA decreased the 45Ca radioactivity in the dialysate, whereas Hcy induced the release of 45Ca. The latter effect was potentiated by glycine, inhibited by the antagonist of group I metabotropic glutamate receptors (mGluR) LY367385, and mimicked by t-ADA, an agonist of these receptors. Electron microscopic examination of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of the microdialysis probe after NMDA application demonstrated swelling of mitochondria, which was prevented by cyclosporin A. This study shows, for the first time, Hcy-induced activation of both group I mGluR and NMDA receptors, which may play a role in acute Hcy neurotoxicity. We present new applications of brain microdialysis in studies on excitotoxicity and neuroprotection.

Abnormal proteolytic degradation of the beta amyloid precursor protein (beta-APP) may result in accumulation of potentially neurotoxic beta amyloid (betaA). The role of various receptors in the regulation of beta-APP processing has been suggested. This study aimed to determine how NMDA receptors and Ca2+ ions regulate proteolysis of beta-APP in rat hippocampus in vitro. Adult rat hippocampal slices were superfused with NMDA-containing media, and immunoreactivity of soluble beta-APP derivatives was detected in dialysates. Application of 100 microM and 250 microM NMDA for 20 min in Ca2+-containing medium induced dose-dependent release of aminoterminal beta-APP derivatives, and a fragment of Abeta sequence, whereas carboxy-terminal fragments of beta-APP were only slightly detected. This indicates activation of beta-APP processing, and release of its soluble cleavage products. This effect was inhibited by NMDA receptor antagonist 1 microM MK-801 and 100 microM CPP in Ca2+-free medium, thus indicating that NMDA receptors and calcium ions mediate proteolytic non-amyloidogenic degradation of the beta-APP.

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.

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.

It is not clear what therapeutic application is most likely for agents blocking glycine site of the NMDA receptors (glycineB). Majority of the studies to date used either glycineB antagonists with doubtful brain penetration or partial agonists. Following systemic administration to rats of our newly developed glycineB antagonists (MRZ 2/570; 2/571 and 2/576) and L-701,324 (MSD) as a reference agent the following behavioural effects were observed: weak (if any) antiparkinsonian-like effects, lack of anxiolytic activity, inhibition of physical and motivational aspects of morphine dependence and neuroprotective activity in global ischaemia. The side effects include: sedation, ataxia, and myorelaxation. We detected neither vacuolisation in the cingulate cortex nor impairment of pre-pulse inhibition indicating lack of psychotomimetic potential.

The role of Ca2+ in neurotransmission and intraneuronal signal transduction under normal conditions, and its involvement in acute and chronic neurodegenerations has been presented in detail in many recent reviews (1–3). Regulatory and neurotoxic increases in intracellular Ca2+ concentration ([Ca2+]i) may result from influx of extracellular Ca2+ to neurones, which in vivo leads to corresponding decrease in extracellular Ca2+ concentration ([Ca2+]e) and/or from its mobilisation from intracellular stores. Failure of Ca2+ extrusion from the cell via ATP-driven Ca2+ pump and the Na+/Ca2+ antiporter may also participate in this process (4, 5).