Group II Metabotropic Glutamate Receptors Reduce Apoptosis and Regulate BDNF and GDNF Levels in Hypoxic-Ischemic Injury in Neonatal Rats.

Birth asphyxia causes brain injury in neonates, but a fully successful treatment has yet to be developed. This study aimed to investigate the effect of group II mGlu receptors activation after experimental birth asphyxia (hypoxia-ischemia) on the expression of factors involved in apoptosis and neuroprotective neurotrophins. Hypoxia-ischemia (HI) on 7-day-old rats was used as an experimental model. The effects of intraperitoneal application of mGluR2 agonist LY379268 (5 mg/kg) and the specific mGluR3 agonist NAAG (5 mg/kg) (1 h or 6 h after HI) on apoptotic processes and initiation of the neuroprotective mechanism were investigated. LY379268 and NAAG applied shortly after HI prevented brain damage and significantly decreased pro-apoptotic Bax and HtrA2/Omi expression, increasing expression of anti-apoptotic Bcl-2. NAAG or LY379268 applied at both times also decreased HIF-1α formation. HI caused a significant decrease in BDNF concentration, which was restored after LY379268 or NAAG administration. HI-induced increase in GDNF concentration was decreased after administration of LY379268 or NAAG. Our results show that activation of mGluR2/3 receptors shortly after HI prevents brain damage by the inhibition of excessive glutamate release and apoptotic damage decrease. mGluR2 and mGluR3 agonists produced comparable results, indicating that both receptors may be a potential target for early treatment in neonatal HI.

The involvement of TRP channels in memory formation and task retrieval in a passive avoidance task in one-day old chicks.

An increase in the intracellular Ca2+ level in neurons is one of the main steps in the memory formation cascade. The increase results from extracellular Ca2+ influx by activation of ionotropic glutamate receptors and release from intracellular stores by the stimulation of IP3 receptors (IP3Rs) via group I metabotropic glutamate receptors (mGluR1/5). Recent data indicate an additional mechanism resulting in Ca2+ influx into neurons, triggered by intracellular signals that are directly connected to the activation of group I mGluRs. This influx occurs through transient receptor potential (TRP) channels, which are permeable to Na+, K+ and, mainly, Ca2+. These channels are activated by increases in intracellular Ca2+, diacylglycerol (DAC) and inositol 1,4,5-triphosphate (IP3) level resulting from a group I mGluR activation. The aim of the present study was to investigate the participation of TRP channels, especially from TRPC and TRPV groups, in memory consolidation and reconsolidation and memory retrieval processes in a passive avoidance task in one-day old chicks. TRP channels were blocked by the injection of the unspecific channel modulators SKF 96365 (2.5 µl 30 µM/hemisphere) and 2-APB (2.5 µl 10 µM/hemisphere) directly into the intermediate medial mesopallium (IMM) region of the chick brain immediately after initial training or after a reminder. The inhibition of specific TRP channels (TRPV1, TRPV3 or TRPC3) was achieved by the application of selective antibodies. Our results demonstrate that the inhibition of TRP channels by the application of both modulators disrupted memory consolidation, resulting in permanent task amnesia. The inhibition of the TRPV1, TRPC3 and TRPV3 channels by specific antibodies resulted in similar amnesia. Moreover, the inhibition of TRP channels by SKF 96365 and 2-APB at different time points after initial training or after the reminder also resulted in amnesia, indicating the role of TRP channels in memory retrieval. The inhibition of calcium influx through these channels resulted in permanent memory disruption, which suggests that the calcium signal generated by TRP channels is crucial for memory formation and retrieval processes. For the first time, the important role of TRPV3 channels in memory formation was demonstrated.

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.

Hypobaric hypoxia postconditioning reduces brain damage and improves antioxidative defense in the model of birth asphyxia in 7-day-old rats.

Perinatal brain insult mostly resulting from hypoxia-ischemia (H-I) often brings lifelong permanent disability, which has a major impact on the life of individuals and their families. The lack of progress in clinically-applicable neuroprotective strategies for birth asphyxia has led to an increasing interest in alternative methods of therapy, including induction of brain tolerance by pre- and particularly postconditioning. Hypoxic postconditioning represents a promising strategy for preventing ischemic brain damage. The aim of this study was to investigate the potential neuroprotective effect of hypobaric hypoxia (HH) postconditioning applied to 7-day old rats after H-I insult. The mild hypobaric conditions (0.47 atm) used in this study imitate an altitude of 5,000 m. We show that application of mild hypobaric hypoxia at relatively short time intervals (1-6 h) after H-I, repeated for two following days leads to significant neuroprotection, manifested by a reduction in weight loss of the ipsilateral hemisphere observed 14 days after H-I. HH postconditioning results in decrease in reactive oxygen species level observed in all experimental groups. The increase in superoxide dismutase activity observed after H-I is additionally enhanced by HH postconditioning applied 1 h after H-I. The increase observed 3 and 6 h after H-I was not statistically significant. Postconditioning with HH suppresses the glutathione concentration decrease evoked by H-I and increased glutathione peroxidase activity and this effect is not dependent on the time of postconditioning initiation. HH postconditioning had no effect on catalase activity. We show for the first time that HH postconditioning reduces brain damage resulting from H-I in immature rats and that the mechanism potentially involved in this effect is related to antioxidant defense mechanisms of immature brain.

Hyperbaric oxygen therapy for the treatment of hypoxic/ischemic injury upon perinatal asphyxia-are we there yet?

Birth asphyxia and its main sequel, hypoxic-ischemic encephalopathy, are one of the leading causes of children's deaths worldwide and can potentially worsen the quality of life in subsequent years. Despite extensive research efforts, efficient therapy against the consequences of hypoxia-ischemia occurring in the perinatal period of life is still lacking. The use of hyperbaric oxygen, improving such vital consequences of birth asphyxia as lowered partial oxygen pressure in tissue, apoptosis of neuronal cells, and impaired angiogenesis, is a promising approach. This review focused on the selected aspects of mainly experimental hyperbaric oxygen therapy. The therapeutic window for the treatment of perinatal asphyxia is very narrow, but administering hyperbaric oxygen within those days improves outcomes. Several miRNAs (e.g., mir-107) mediate the therapeutic effect of hyperbaric oxygen by modulating the Wnt pathway, inhibiting apoptosis, increasing angiogenesis, or inducing neural stem cells. Combining hyperbaric oxygen therapy with drugs, such as memantine or ephedrine, produced promising results. A separate aspect is the use of preconditioning with hyperbaric oxygen. Overall, preliminary clinical trials with hyperbaric oxygen therapy used in perinatal asphyxia give auspicious results.

Hyperbaric oxygen and hyperbaric air treatment result in comparable neuronal death reduction and improved behavioral outcome after transient forebrain ischemia in the gerbil.

Anoxic brain injury resulting from cardiac arrest is responsible for approximately two-thirds of deaths. Recent evidence suggests that increased oxygen delivered to the brain after cardiac arrest may be an important factor in preventing neuronal damage, resulting in an interest in hyperbaric oxygen (HBO) therapy. Interestingly, increased oxygen supply may be also reached by application of normobaric oxygen (NBO) or hyperbaric air (HBA). However, previous research also showed that the beneficial effect of hyperbaric treatment may not directly result from increased oxygen supply, leading to the conclusion that the mechanism of hyperbaric prevention of brain damage is not well understood. The aim of our study was to compare the effects of HBO, HBA and NBO treatment on gerbil brain condition after transient forebrain ischemia, serving as a model of cardiac arrest. Thereby, we investigated the effects of repetitive HBO, HBA and NBO treatment on hippocampal CA1 neuronal survival, brain temperature and gerbils behavior (the nest building), depending on the time of initiation of the therapy (1, 3 and 6 h after ischemia). HBO and HBA applied 1, 3 and 6 h after ischemia significantly increased neuronal survival and behavioral performance and abolished the ischemia-evoked brain temperature increase. NBO treatment was most effective when applied 1 h after ischemia; later application had a weak or no protective effect. The results show that HBO and HBA applied between 1 and 6 h after ischemia prevent ischemia-evoked neuronal damage, which may be due to the inhibition of brain temperature increase, as a result of the applied rise in ambient pressure, and just not due to the oxygen per se. This perspective is supported by the finding that NBO treatment was less effective than HBO or HBA therapy. The results presented in this paper may pave the way for future experimental studies dealing with pressure and temperature regulation.

Differential involvement of mGluR1 and mGluR5 in memory reconsolidation and retrieval in a passive avoidance task in 1-day old chicks.

Group I metabotropic glutamate receptors (mGluRs) are involved in memory formation. The Ca2+ signal derived from stimulation of IP3 receptors (IP3Rs) via mGluRs, initiates protein synthesis that is required for memory consolidation and reconsolidation. However it has been suggested that different mechanisms are triggered by mGluR1/5 activation in these two processes. It is also not clear whether the transient amnesia observed after blockade of group I mGluRs after a reminder, results from disturbance of memory reconsolidation or temporal impairment of recall. The aim of this study was to examine more closely the role of mGluR1 in memory consolidation and reconsolidation and to detect differences in the participation of mGluR1 and mGluR5 in memory retrieval after initial training and after the remainder of the task. Our results demonstrate, that in chicks performing a one-trial passive avoidance task, antagonists of mGluR1, mGluR5 and IP3R significantly disturb memory consolidation and reconsolidation. Inhibition of mGluR5 and IP3R also impairs memory recall, whereas mGluR1 do not seem to participate in this process. The presented data suggest that activation of mGluR1 and mGluR5 is necessary for the correct course of memory consolidation and reconsolidation, whereas mGluR5 are additionally involved in retrieval processes dependent on Ca2+ release from IP3 activated intracellular stores.

[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.

Pretreatment with mGluR2 or mGluR3 Agonists Reduces Apoptosis Induced by Hypoxia-Ischemia in Neonatal Rat Brains.

Hypoxia-ischemia (HI) in an immature brain results in energy depletion and excessive glutamate release resulting in excitotoxicity and oxidative stress. An increase in reactive oxygen species (ROS) production induces apoptotic processes resulting in neuronal death. Activation of group II mGluR was shown to prevent neuronal damage after HI. The application of agonists of mGluR3 (N-acetylaspartylglutamate; NAAG) or mGluR2 (LY379268) inhibits the release of glutamate and reduces neurodegeneration in a neonatal rat model of HI, although the exact mechanism is not fully recognized. In the present study, the effects of NAAG (5 mg/kg) and LY379268 (5 mg/kg) application (24 h or 1 h before experimental birth asphyxia) on apoptotic processes as the potential mechanism of neuroprotection in 7-day-old rats were investigated. Intraperitoneal application of NAAG or LY379268 at either time point before HI significantly reduced the number of TUNEL-positive cells in the CA1 region of the ischemic brain hemisphere. Both agonists reduced expression of the proapoptotic Bax protein and increased expression of Bcl-2. Decreases in HI-induced caspase-9 and caspase-3 activity were also observed. Application of NAAG or LY379268 24 h or 1 h before HI reduced HIF-1α formation likely by reducing ROS levels. It was shown that LY379268 concentration remains at a level that is required for activation of mGluR2 for up to 24 h; however, NAAG is quickly metabolized by glutamate carboxypeptidase II (GCPII) into glutamate and N-acetyl-aspartate. The observed effect of LY379268 application 24 h or 1 h before HI is connected with direct activation of mGluR2 and inhibition of glutamate release. Based on the data presented in this study and on our previous findings, we conclude that the neuroprotective effect of NAAG applied 1 h before HI is most likely the result of a combination of mGluR3 and NMDA receptor activation, whereas the beneficial effects of NAAG pretreatment 24 h before HI can be explained by the activation of NMDA receptors and induction of the antioxidative/antiapoptotic defense system triggered by mild excitotoxicity in neurons. This response to NAAG pretreatment is consistent with the commonly accepted mechanism of preconditioning.

The Mechanism of the Neuroprotective Effect of Kynurenic Acid in the Experimental Model of Neonatal Hypoxia-Ischemia: The Link to Oxidative Stress.

The over-activation of NMDA receptors and oxidative stress are important components of neonatal hypoxia-ischemia (HI). Kynurenic acid (KYNA) acts as an NMDA receptor antagonist and is known as a reactive oxygen species (ROS) scavenger, which makes it a potential therapeutic compound. This study aimed to establish the neuroprotective and antioxidant potential of KYNA in an experimental model of HI. HI on seven-day-old rats was used as an experimental model. The animals were injected i.p. with different doses of KYNA 1 h or 6 h after HI. The neuroprotective effect of KYNA was determined by the measurement of brain damage and elements of oxidative stress (ROS and glutathione (GSH) level, SOD, GPx, and catalase activity). KYNA applied 1 h after HI significantly reduced weight loss of the ischemic hemisphere, and prevented neuronal loss in the hippocampus and cortex. KYNA significantly reduced HI-increased ROS, GSH level, and antioxidant enzyme activity. Only the highest used concentration of KYNA showed neuroprotection when applied 6 h after HI. The presented results indicate induction of neuroprotection at the ROS formation stage. However, based on the presented data, it is not possible to pinpoint whether NMDA receptor inhibition or the scavenging abilities are the dominant KYNA-mediated neuroprotective mechanisms.

N-Acetylaspartylglutamate (NAAG) Pretreatment Reduces Hypoxic-Ischemic Brain Damage and Oxidative Stress in Neonatal Rats.

N-acetylaspartylglutamate (NAAG), the most abundant peptide transmitter in the mammalian nervous system, activates mGluR3 at presynaptic sites, inhibiting the release of glutamate, and acts on mGluR3 on astrocytes, stimulating the release of neuroprotective growth factors (TGF-ß). NAAG can also affect N-methyl-d-aspartate (NMDA) receptors in both synaptic and extrasynaptic regions. NAAG reduces neurodegeneration in a neonatal rat model of hypoxia-ischemia (HI), although the exact mechanism is not fully recognized. In the present study, the effect of NAAG application 24 or 1 h before experimental birth asphyxia on oxidative stress markers and the potential mechanisms of neuroprotection on 7-day old rats was investigated. The intraperitoneal application of NAAG at either time point before HI significantly reduced the weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content and activity of antioxidant enzymes, and increased the concentration of reduced glutathione (GSH). No additional increase in the TGF-ß concentration was observed after NAAG application. The fast metabolism of NAAG and the decrease in TGF-ß concentration that resulted from NAAG pretreatment, performed up to 24 h before HI, excluded the involvement mGluR3 in neuroprotection. The observed effect may be explained by the activation of NMDA receptors induced by NAAG pretreatment 24 h before HI. Inhibition of the NAAG effect by memantine supports this conclusion. NAAG preconditioning 1 h before HI results in a mixture of mGluR3 and NMDA receptor activation. Preconditioning with NAAG induces the antioxidative defense system triggered by mild excitotoxicity in neurons. Moreover, this response to NAAG pretreatment is consistent with the commonly accepted mechanism of preconditioning. However, this theory requires further investigation.

Hypoxic Roadmap of Glioblastoma-Learning about Directions and Distances in the Brain Tumor Environment.

Malignant brain tumor-glioblastoma is not only difficult to treat but also hard to study and model. One of the reasons for these is their heterogeneity, i.e., individual tumors consisting of cancer cells that are unlike each other. Such diverse cells can thrive due to the simultaneous co-evolution of anatomic niches and adaption into zones with distorted homeostasis of oxygen. It dampens cytotoxic and immune therapies as the response depends on the cellular composition and its adaptation to hypoxia. We explored what transcriptome reposition strategies are used by cells in the different areas of the tumor. We created the hypoxic map by differential expression analysis between hypoxic and cellular features using RNA sequencing data cross-referenced with the tumor's anatomic features (Ivy Glioblastoma Atlas Project). The molecular functions of genes differentially expressed in the hypoxic regions were analyzed by a systematic review of the gene ontology analysis. To put a hypoxic niche signature into a clinical context, we associated the model with patients' survival datasets (The Cancer Genome Atlas). The most unique class of genes in the hypoxic area of the tumor was associated with the process of autophagy. Both hypoxic and cellular anatomic features were enriched in immune response genes whose, along with autophagy cluster genes, had the power to predict glioblastoma patient survival. Our analysis revealed that transcriptome responsive to hypoxia predicted worse patients' outcomes by driving tumor cell adaptation to metabolic stress and immune escape.

MicroRNA in Brain pathology: Neurodegeneration the Other Side of the Brain Cancer.

The mammalian brain is made up of billions of neurons and supporting cells (glial cells), intricately connected. Molecular perturbations often lead to neurodegeneration by progressive loss of structure and malfunction of neurons, including their death. On the other side, a combination of genetic and cellular factors in glial cells, and less frequently in neurons, drive oncogenic transformation. In both situations, microenvironmental niches influence the progression of diseases and therapeutic responses. Dynamic changes that occur in cellular transcriptomes during the progression of developmental lineages and pathogenesis are controlled through a variety of regulatory networks. These include epigenetic modifications, signaling pathways, and transcriptional and post-transcriptional mechanisms. One prominent component of the latter is small non-coding RNAs, including microRNAs, that control the vast majority of these networks including genes regulating neural stemness, differentiation, apoptosis, projection fates, migration and many others. These cellular processes are also profoundly dependent on the microenvironment, stemness niche, hypoxic microenvironment, and interactions with associated cells including endothelial and immune cells. Significantly, the brain of all other mammalian organs expresses the highest number of microRNAs, with an additional gain in expression in the early stage of neurodegeneration and loss in expression in oncogenesis. However, a mechanistic explanation of the concept of an apparent inverse correlation between the odds of cancer and neurodegenerative diseases is only weakly developed. In this review, we thus will discuss widespread de-regulation of microRNAome observed in these two major groups of brain pathologies. The deciphering of these intricacies is of importance, as therapeutic restoration of pre-pathological microRNA landscape in neurodegeneration must not lead to oncogenesis and vice versa. We thus focus on microRNAs engaged in cellular processes that are inversely regulated in these diseases. We also aim to define the difference in microRNA networks between pro-survival and pro-apoptotic signaling in the brain.

MicroRNA-451 Inhibits Migration of Glioblastoma while Making It More Susceptible to Conventional Therapy.

Malignant glioblastoma (GBM, glioma) is the most common and aggressive primary adult brain tumor. The prognosis of GBM patients remains poor, despite surgery, radiation and chemotherapy. The major obstacles for successful remedy are invasiveness and therapy resistance of GBM cells. Invasive glioma cells leave primary tumor core and infiltrate surrounding normal brain leading to inevitable recurrence, even after surgical resection, radiation and chemotherapy. Therapy resistance allowing for selection of more aggressive and resistant sub-populations including GBM stem-like cells (GSCs) upon treatment is another serious impediment to successful treatment. Through their regulation of multiple genes, microRNAs can orchestrate complex programs of gene expression and act as master regulators of cellular processes. MicroRNA-based therapeutics could thus impact broad cellular programs, leading to inhibition of invasion and sensitization to radio/chemotherapy. Our data show that miR-451 attenuates glioma cell migration in vitro and invasion in vivo. In addition, we have found that miR-451 sensitizes glioma cells to conventional chemo- and radio-therapy. Our data also show that miR-451 is regulated in vivo by AMPK pathway and that AMPK/miR-451 loop has the ability to switch between proliferative and migratory pattern of glioma cells behavior. We therefore postulate that AMPK/miR-451 negative reciprocal feedback loop allows GBM cells/GSCs to adapt to tumor "ecosystem" by metabolic and behavioral flexibility, and that disruption of such a loop reduces invasiveness and diminishes therapy resistance.

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.

Calcium ions in neuronal degeneration.

Neuronal Ca(2+) homeostasis and Ca(2+) signaling regulate multiple neuronal functions, including synaptic transmission, plasticity, and cell survival. Therefore disturbances in Ca(2+) homeostasis can affect the well-being of the neuron in different ways and to various degrees. Ca(2+) homeostasis undergoes subtle dysregulation in the physiological ageing. Products of energy metabolism accumulating with age together with oxidative stress gradually impair Ca(2+) homeostasis, making neurons more vulnerable to additional stress which, in turn, can lead to neuronal degeneration. Neurodegenerative diseases related to aging, such as Alzheimer's disease, Parkinson's disease, or Huntington's disease, develop slowly and are characterized by the positive feedback between Ca(2+) dyshomeostasis and the aggregation of disease-related proteins such as amyloid beta, alfa-synuclein, or huntingtin. Ca(2+) dyshomeostasis escalates with time eventually leading to neuronal loss. Ca(2+) dyshomeostasis in these chronic pathologies comprises mitochondrial and endoplasmic reticulum dysfunction, Ca(2+) buffering impairment, glutamate excitotoxicity and alterations in Ca(2+) entry routes into neurons. Similar changes have been described in a group of multifactorial diseases not related to ageing, such as epilepsy, schizophrenia, amyotrophic lateral sclerosis, or glaucoma. Dysregulation of Ca(2+) homeostasis caused by HIV infection or by sudden accidents, such as brain stroke or traumatic brain injury, leads to rapid neuronal death. The differences between the distinct types of Ca(2+) dyshomeostasis underlying neuronal degeneration in various types of pathologies are not clear. Questions that should be addressed concern the sequence of pathogenic events in an affected neuron and the pattern of progressive degeneration in the brain itself. Moreover, elucidation of the selective vulnerability of various types of neurons affected in the diseases described here will require identification of differences in the types of Ca(2+) homeostasis and signaling among these neurons. This information will be required for improved targeting of Ca(2+) homeostasis and signaling components in future therapeutic strategies, since no effective treatment is currently available to prevent neuronal degeneration in any of the pathologies described here.

Dantrolene antagonizes the glycineB site of the NMDA receptor.

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.

Neuroprotective effects of nicotinamide and 1-methylnicotinamide in acute excitotoxicity in vitro.

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.

Pretreatment with Group II Metabotropic Glutamate Receptor Agonist LY379268 Protects Neonatal Rat Brains from Oxidative Stress in an Experimental Model of Birth Asphyxia.

Hypoxia-ischemia (H-I) at the time of birth may cause neonatal death or lead to persistent brain damage. The search for an effective treatment of asphyxiated infants has not resulted in an effective therapy, and hypothermia remains the only available therapeutic strategy. Among possible experimental therapies, the induction of ischemic tolerance is promising. Recent investigations have shown that activation of group II metabotropic glutamate receptors (mGluR2/3) can provide neuroprotection against H-I, but the mechanism of this effect is not clear. The aim of this study was to investigate whether an mGluR2/3 agonist applied before H-I reduces brain damage in an experimental model of birth asphyxia and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I on seven-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intraperitoneally with the mGluR2/3 agonist LY379268 24 or 1 h before H-I (5 mg/kg). LY379268 reduced the infarct area in the ischemic hemisphere. Application of the agonist at both times also reduced the elevated levels of reactive oxygen species (ROS) in the ipsilateral hemisphere observed after H-I and prevented the increase in antioxidant enzyme activity in the injured hemisphere. The decrease in glutathione (GSH) level was also restored after agonist application. The results suggest that the neuroprotective mechanisms triggered by the activation of mGluR2/3 before H-I act through the decrease of glutamate release and its extracellular concentration resulting in the inhibition of ROS production and reduction of oxidative stress. This, rather than induction of ischemic tolerance, is probably the main mechanism involved in the observed neuroprotection.