Tractography passes the test: Results from the diffusion-simulated connectivity (disco) challenge.

Estimating structural connectivity from diffusion-weighted magnetic resonance imaging is a challenging task, partly due to the presence of false-positive connections and the misestimation of connection weights. Building on previous efforts, the MICCAI-CDMRI Diffusion-Simulated Connectivity (DiSCo) challenge was carried out to evaluate state-of-the-art connectivity methods using novel large-scale numerical phantoms. The diffusion signal for the phantoms was obtained from Monte Carlo simulations. The results of the challenge suggest that methods selected by the 14 teams participating in the challenge can provide high correlations between estimated and ground-truth connectivity weights, in complex numerical environments. Additionally, the methods used by the participating teams were able to accurately identify the binary connectivity of the numerical dataset. However, specific false positive and false negative connections were consistently estimated across all methods. Although the challenge dataset doesn't capture the complexity of a real brain, it provided unique data with known macrostructure and microstructure ground-truth properties to facilitate the development of connectivity estimation methods.

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.

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.

The role of the glutamatergic NMDA receptor in nanosilver-evoked neurotoxicity in primary cultures of cerebellar granule cells.

Nanoparticles are known to enter the vertebrate brain, but little is known about their neurotoxicity. The aim of this study is to investigate mechanisms of the contribution of AgNPs to neuronal cell death using primary cultures of rat cerebellar granule cells (CGCs). We tested the role of glutamatergic N-methyl-d-aspartate receptors (NMDA) in AgNP-evoked neurotoxicity using MK-801, a noncompetitive inhibitor of NMDAR. We used commercially available 0.2% PVP-coated AgNPs <100 nm in a concentration range of 2.5-75 µg/ml sonicated with fetal calf serum. After a 10 min incubation period, a dose-dependent increase in the uptake of (45)Ca(2+) into neurons was observed in the presence of 25-75 µg/mL AgNPs which was completely abolished by addition of MK-801. Using the fluorescent dye fluo3 AM we observed an increase in the intracellular calcium level by 87% compared to control. ROS production was found to increase by about 30% over control after a 30-min incubation with 75 µg/mL AgNPs. Further, we observed a significant decrease in the mitochondrial potential during a 30-min incubation with AgNPs. Administration of MK-801 was found to provide a protective effect. Our results show that excitotoxicity via activation of NMDA receptor, followed by calcium imbalance, destabilization of mitochondrial function and ROS production, indicate an important mechanism involved in neurotoxicity evoked by AgNPs in cultured neurons.

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.

Excitotoxic neuronal injury in acute homocysteine neurotoxicity: role of calcium and mitochondrial alterations.

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 1mM 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.

Synthetic bastadins modify the activity of ryanodine receptors in cultured cerebellar granule cells.

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 concentrations >20 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.

Effects of methionine sulfoximine on the glutamine and glutamate content and cell volume in rat cerebral cortical slices: involvement of mechanisms not related to inhibition of glutamine synthesis.

Treatment of cerebral cortical slices with 5mM ammonium acetate (ammonia) elevated the glutamine (Gln) content and increased cell volume in the slices, in agreement with the postulated contribution of glutamine to hyperammonemic brain edema [Neurochem. Int. 43 (2003) 299]. In this study we show that, unexpectedly, treatment with a glutamine synthetase inhibitor, methionine sulfoximine (MSO) (0.1-5.0mM) in the absence of ammonia increases Gln content in the slices in a dose-independent manner, to levels higher than those recorded after ammonia treatment. MSO (>0.1mM) inhibited (>0.1mM) Gln uptake in crude cerebral cortical cell membranes (P2 fraction). Since Gln uptake in this preparation was largely facilitated by the Gln efflux-promoting systems ASC and N and less so by the uptake promoting system A, MSO-induced accumulation of Gln could result from inhibition of Gln efflux. MSO did not affect cell volume in the slices, showing that Gln retention is not as a rule a causative factor in cerebral edema. MSO at 5mM concentration increased cell swelling induced by ammonia, which is consistent with earlier observations pointing to the direct excitotoxic action of MSO in vivo and in vitro. The results emphasize the limits of applicability of MSO as an inhibitor of Gln synthesis in an in vitro system.

Plasma homocysteine level and the course of ischemic stroke.

Increased level of homocysteine (Hcy) in blood seems to influence negatively the course of ischemic stroke (IS), the possible mechanism of this action could be acceleration of oxidative stress. The aim of this study is to assess the influence of Hcy level in patients with IS on the prognosis 3 months after the stroke onset. 75 patients aged 68.27 +/- 12.62 years, with the diagnosis of first ever IS were examined. Patients with the symptoms corresponding with TACS at the beginning of stroke and with diminished level of consciousness were not included. The level of Hcy over 15 micromol/l was assessed as mild hiperhomocysteinemia (MHcy). 74 (98.7%) patients were assessed 3 months after IS onset in the Rankin scale. Recovery was assessed, according to Rankin Scale: good recovery (GR) 0-2, bad recovery (BR) 3-5 and death. MHcy was seen in 9 (14.5%) with GR and in 8 (66.7%) with BR (P = 0.0005). MHcy increases the risk of BR 11.78 times (95% CI 2.93-47.42).

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones.

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg(2+)-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC(50) of 17.4mM, whereas EC(50) for L-glutamate was 0.17 mM. Addition of 50 microM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 microM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25 mM D,L-homocysteine with 50 microM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 microM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca(2+) to neurones.

Metabotropic glutamate receptors (mGluRs) are involved in early phase of memory formation: possible role of modulation of glutamate release.

Metabotropic glutamate receptors (mGluRs) groups I and II are involved in the cellular processes of long-term potentiation (LTP) and learning and memory formation. I.c.v. injection of the mGluRs agonist 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) can impair memory formation in some types of learning task. The role of mGluRs in neurotransmitters release and production of second messengers has been suggested. The aim of the present study was to determine the effect of i.c.v. administration of the new potent mGluRs agonist ABHxD-I and compare its effect with that of ACPD. We studied the effect of both agonists on acquisition and memory for a one-trial passive avoidance learning task in day-old chicks and on the training related glutamate (Glu) release. ACPD or ABHxD-I (50 nmole per chick, i.c.v. injection) were administered at different times before or after training and chicks were tested at various times after training. Chicks injected with ABHxD-I 30 min before training showed amnesia when tested 30 min or 3h after training. The amnestic effect of ACPD was significant only 30 min after training. Glu release evoked by 70 mM KCl was measured in slices prepared from the IMHV of chick brain isolated from animals injected with either ACPD or ABHxD-I 30 min before training and tested 30 min after training. Glu concentration was measured using HPLC. Both ACPD and ABHxD-I significantly increased Glu release in slices isolated from untrained chicks (30 and 48% compare to control, respectively, P<0.05). Training itself increased Glu release (41% compared to control, P<0.01) and no additional effect of either ACPD or ABHxD-I was observed. These results suggest that mGluRs groups I and II are involved in the early stages of memory formation and that application of either of the studied mGluRs agonists may interfere with that process. The amnestic effect of ABHxD-I seems to be stronger and longer lasting. Although the mechanism of this effect still remains unclear, our results suggest that disregulation of Glu release by mGluR agonists may participate in this process.

Homocysteine-evoked 45Ca release in the rabbit hippocampus is mediated by both NMDA and group I metabotropic glutamate receptors: in vivo microdialysis study.

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.