Zinc and Copper Brain Levels and Expression of Neurotransmitter Receptors in Two Rat ASD Models.

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.

Changes in the Expression of SNAP-25 Protein in the Brain of Juvenile Rats in Two Models of Autism.

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.

Altered expression of glutamatergic and GABAergic genes in the valproic acid-induced rat model of autism: A screening test.

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.

Tetrabromobisphenol A-induced depolarization of rat cerebellar granule cells: ex vivo and in vitro studies.

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.

Altered expression of genes involved in programmed cell death in primary cultured rat cerebellar granule cells acutely challenged with tetrabromobisphenol A.

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.

Tetrabromobisphenol A disturbs zinc homeostasis in cultured cerebellar granule cells: A dual role in neurotoxicity.

The brominated flame retardant tetrabromobisphenol A (TBBPA) has recognized neurotoxic properties mediated by intracellular Ca2+ imbalance and oxidative stress. Although these factors are known to trigger the release of Zn2+ from intracellular stores, the effects of TBBPA on Zn2+ homeostasis in neurons and the role of Zn2+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 Zn2+ from the intracellular stores and increases its intracellular concentration, followed by Zn2+ displacement from the cells. TBBPA-evoked Zn2+ transients are partially mediated by Ca2+ and ROS. Application of TPEN, Zn2+ chelator, potentiates TBBPA- and glutamate-induced 45Ca 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 Zn2+. 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 Zn2+ homeostasis in neuronal cultures, and we revealed complex roles for endogenous Zn2+ in cytoprotection and TBBPA toxicity in cultured neurons.

The Role of Ca2+ Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A.

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.

Select putative neurodevelopmental toxins modify SNAP-25 expression in primary cultures of rat cerebellar granule cells.

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 24h. 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.

Hypobaric Preconditioning Modifies Group I mGluRs Signaling in Brain Cortex.

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 Cß1 (PLCß1), which is a major mediator of mGluR1/5 signaling. Immunocytochemical analysis of the cortex revealed increase in the mGluR1/5 and PLCß1 IR in perikarya, and a decrease in IR of the neuronal inositol trisphosphate receptors (IP3Rs). We suggest that enhanced expression of mGluR5 and PLCß1 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.

Role of Ryanodine and NMDA Receptors in Tetrabromobisphenol A-Induced Calcium Imbalance and Cytotoxicity in Primary Cultures of Rat Cerebellar Granule Cells.

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.

Antidepressant-like and anxiolytic-like effects of mild hypobaric hypoxia in mice: possible involvement of neuropeptide Y.

Previous studies have demonstrated that repeated submission of rats to mild hypobaric hypoxia reduces the persistent behavioral and hormonal depressive symptoms induced by exposure to footshock in the learned helplessness paradigm. The aim of this study was to determine whether hypoxic preconditioning of mice can also induce antidepressant- and anxiolytic-like effects that are detectable with the other commonly used behavioral tests, and to determine whether these effects are accompanied by an increase in neuropeptide Y (NPY) in the hippocampus, which may suggest the involvement of NPY in these mechanisms. The intermittent mild hypobaric hypoxia was generated by 2-h exposure of mice to 0.47 atm for 3 consecutive days. In the tail suspension test a significant decrease in the duration of immobility was observed 24 h, but not 48 h after the last hypobaric session. The elevated plus maze trials performed 48 h after preconditioning showed a significant increase in the frequency of open arm entries, a reduction in the duration of closed arm occupancy and substantially more time spent in the open arms in comparison to the control groups. The open field test demonstrated the absence of increases in general activity or unspecific exploratory behavior in hypoxia-preconditioned mice. The EIA test detected a statistically significant but relatively weak increase in the NPY content in the hippocampus 24 h after preconditioning. Together, our data demonstrate that preconditioning of mice with intermittent mild hypobaric hypoxia induces anxiolytic- and antidepressant-like effects. They are accompanied by up-regulation of NPY which may suggest its mechanistic role.

Effects of mGluR5 positive and negative allosteric modulators on brain damage evoked by hypoxia-ischemia in neonatal rats.

In the present study, we examined the effects of negative and positive allosteric modulators of metabotropic glutamate receptor 5 (mGluR5), fenobam and ADX47273, respectively, on brain damage induced by hypoxia-ischemia (H-I) in 7-day-old rats. The test drugs were administered intraperitoneally 10 min after H-I. Rectal body temperature was measured for 2.5 h after the insult. The number of apoptotic neurons in the immature rat brain was evaluated after 24 h. The wet weight of both hemispheres was determined 14 days after H-I, and its loss was used as an indicator of brain damage. In the vehicle-treated groups, H-I reduced the weight of the ipsilateral (ischemic) hemisphere by approximately 33% and sixfold increased the number of apoptotic cells in the cortex. Fenobam (10 mg/kg) and ADX47273 (5, 10, and 30 mg/kg) had no significant effect on brain damage, although application of fenobam at this dose significantly reduced the number of apoptotic cells. In contrast, fenobam (20 mg/kg) potentiated ischemic brain damage to 57.4% and had no effect on H-I-induced apoptosis. In all of the experimental groups, we detected no significant changes in the weight of the contralateral (control) hemisphere or the rectal temperature. In conclusion, in 7-day-old rats, the bidirectional modulation of mGluR5 by fenobam (10 mg/kg) and ADX47273 (all doses tested) did not result in significant changes in H-I-evoked brain damage, supporting our previous data indicating that also the antagonists of mGluR5 MPEP and MTEP, which reduce neuronal lesions in adult animals submitted to brain ischemia, were ineffective in 7-day-old rat pups.

Delayed preconditioning with NMDA receptor antagonists in a rat model of perinatal asphyxia.

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.

1MeTIQ provides protection against Aß-induced reduction of surface expression of synaptic proteins and inhibits H2O2-induced oxidative stress in primary hippocampal neurons.

Alzheimer's disease (AD) is associated with increased brain levels of ß-amyloid (Aß) peptides, which readily self-aggregate into fibrils and oligomers that have particularly deleterious properties toward synapses of excitatory glutamatergic neurons. Here, we examined the neuroprotective effects of 1-methyl-1,2,3,4,-tetrahydroisoquinoline (1MeTIQ) against Aß-induced loss of synaptic proteins in cultured primary hippocampal neurons. Exposure of mature primary hippocampal neurons to 10 µM synthetic Aß1-40 over 72 h resulted in ~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 Aß1-40-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.

Blocking NMDA receptors delays death in rats with acute liver failure by dual protective mechanisms in kidney and brain.

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.

NMDA receptor antagonists MK-801 and memantine induce tolerance to oxygen and glucose deprivation in primary cultures of rat cerebellar granule cells.

Preconditioning is an experimental strategy for reducing ischemic brain damage. There are reports that brief exposure of neurons to NMDA-receptor antagonists may be an adequate preconditioning stressor. We studied effects of preconditioning of the cerebellar granule cells (CGC) in primary culture by 30-minute exposure to NMDA receptor antagonists 0.5 µM MK-801 or 5 µM memantine. CGC were challenged with oxygen and glucose deprivation (OGD) or excitotoxic glutamate and cell viability was tested 24 h later using calcein/ethidium homodimer-1 staining. We studied glutamate-induced increases in 45Ca uptake and in the intracellular Ca2+ level assessed with the fluorescent probe fluo-3. The number of living cells in OGD-treated cultures decreased by 42%. Preconditioning with MK-801 or memantine 24 h earlier reduced cell death to 8% and 30% and 48 h earlier to 27% and 33%, respectively. Pretreatment with MK-801 followed by the standard MK-801 wash out was slightly cytoprotective in a glutamate excitotoxicity test performed immediately; the protection increased significantly 24 h after preconditioning. In both cases the extensive wash out of MK-801 after preconditioning resulted in loss of cytoprotection. The increase in the intracellular Ca2+ level evoked by glutamate was decreased 24 h after preconditioning and even halved in the neuronal cultures 48 h after preconditioning with MK-801 and memantine. Glutamate-induced 45Ca uptake in these cells was decreased by 18%, irrespective of the time laps after preconditioning. These results demonstrate that preconditioning of CGC with NMDA receptor antagonists induces prolonged tolerance to OGD, which is accompanied by the reduction of glutamate-evoked calcium fluxes. The causal relationship between these effects may be suggested.

Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress.

BACKGROUND: Tetrabromobisphenol A (TBBPA) is a toxic brominated flame retardant. Previous studies have demonstrated that exposure of primary cultures of rat cerebellar granule cells (CGC) to ≥ 10 µM TBBPA induces toxicity and excitotoxicity, and the underlying mechanism may involve calcium imbalance and oxidative stress. Here we examined whether the application of TBBPA at subtoxic concentrations may exacerbate acute damage of CGC challenged with oxygen-glucose deprivation (OGD), and evaluated with fluorescent indicators the involvement of calcium imbalance, mitochondrial depolarization and oxidative stress. METHODS: Survival of CGC was assessed 24 h after OGD/TBBPA using fluorescent dyes. An OGD challenge lasting for 45, 60 or 75 min induced a duration-dependent injury to the neurons. RESULTS: Application of 2.5, 5 or 7.5 µM TBBPA for 45 min to normoxic and glucose-containing incubation medium did not reduce the viability of cultured CGC, but this compound exacerbated the toxic effects of OGD in a concentration-dependent way. Moreover, TBBPA had a slight effect on calcium homeostasis and mitochondrial membrane potential, but significantly activated the production of reactive oxygen species in CGC. The application of H(2)O(2) at 5, 10 and 25 µM mimicked the effects of TBBPA on OGD toxicity, while 0.1 mM ascorbic acid or 1 mM glutathione ameliorated this toxicity. CONCLUSION: These results suggest the involvement of oxidative stress in the synergistic neurotoxic effects of TBBPA and OGD.

In vivo hippocampal microdialysis reveals impairment of NMDA receptor-cGMP signaling in APP(SW) and APP(SW)/PS1(L166P) Alzheimer's transgenic mice.

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer's disease (AD) including memory deficit, presence of ß-amyloid (Aß) 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 Aß 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 APP(SW) and 4 months old APP(SW)/PS1(L166P) 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. Aß oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5±3.6ng/g in 4 months old APP(SW) mice to 60.8±15.9ng/g in 4 months old APP(SW)/PS1(L166P) mice. Four months old APP(SW) mice had no Aß amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient Aß 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 Aß oligomers but predating onset of Aß amyloidosis.

Repeated exposure of adult rats to Aroclor 1254 induces neuronal injury and impairs the neurochemical manifestations of the NMDA receptor-mediated intracellular signaling in the hippocampus.

Aroclor 1254 is a mixture of polychlorinated biphenyls (PCBs), a class of environmental toxins which cause a wide spectrum of neurotoxic effects. Learning and memory deficits are the profound effects of PCBs which may be related to hippocampal dysfunction. To get insight into the underlying neurochemical mechanisms, we employed the microdialysis technique to investigate the effect of repeated exposure of adult male Wistar rats to Aroclor 1254 (10mg/kg b.w., daily, ig., for 14days), on the neurochemical parameters of NMDA receptor-mediated glutamatergic signaling in the hippocampus in vivo assessed using the microdialysis technique. The results demonstrated that exposure to Aroclor 1254, which was associated with substantial neuronal damage and loss in the hippocampus, markedly decreased the NMDA-induced extracellular accumulation of newly loaded (45)CaCl(2), cGMP and glutamate, and reduced the basal content of the NO precursor, arginine, indicating inhibition of the NMDA/NO/cGMP pathway. Aroclor 1254 exposure also decreased the basal microdialysate content of glutamate and glutamine, which may cause inadequate supply of the neurotransmitter glutamate, while the level of two other neuroactive amino acids, aspartate or taurine was not affected by the exposure. The results underscore neuronal lesion and inhibition of NMDA receptor-mediated glutamatergic signaling in hippocampus as a potential major contributor to the cognitive deficits associated with exposure to PCB.

[Role of calcium in physiology and pathology of neurons]. / Rola wapnia w fizjologii i patologii neuronów.

In this article we present evolutionary aspects of the dual role of Ca2+ as signaling molecules and cytotoxic cations. We discuss the mechanisms of calcium homeostasis in neurons, taking into account the specific features of excitable cells and the mechanisms of generation and transduction of calcium signals. Based on this information we outline the role of Ca2+ ions in specific functions of the nerve cell, such as excitability, propagation of the action potential, synaptic transmission, neuronal plasticity and various forms of mobility. Then we discuss the role of disturbances of calcium homeostasis and signaling function in the mechanisms of injury and death of neurons in acute diseases with special regard to cerebral ischemia, and in chronic neurodegenerative disorders.