http://repub.eur.nl/res/aut/52441/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/37390/ 2013-01-01T00:00:00Z Hippocampal sclerosis, the main pathological sign of chronic temporal lobe epilepsy (TLE), is associated with oxidative injury, altered N-methyl d-aspartate receptor (NMDAR) stoichiometry, and loss of hippocampal neurons. However, the mechanisms that drive the chronic progression of TLE remain elusive. Our previous studies have shown that NADPH oxidase activation and ERK 1/2 phosphorylation are required for the up-regulation of the predominantly pre-synaptic NR2B subunit auto-receptor in both in vitro and in vivo pilocarpine (PILO) models of TLE. To provide further understanding of the cellular responses during the early-stages of hyper excitability, we investigated the role of oxidative damage and altered NR2B functions. In rat primary hippocampal cultures, we found that N-acetylcysteine (NAC) prevented PILO-mediated thiol oxidation, apoptosis, cell death and NR2B subunit over-expression. Interestingly, NAC did not block thiol oxidation when added to the neurons 6. h after the PILO exposure, suggesting that disulfide formation could rapidly become an irreversible phenomenon. Moreover, NAC pre-treatment did not prevent PILO-induced NR2A subunit over-expression, a critical event in hippocampal sclerosis. Pre-treatment with the highly specific NR2B subunit inhibitor, ifenprodil, partially decreased PILO-mediated thiol oxidation and was not effective in preventing apoptosis and cell death. However, if acutely administered 48. h after PILO exposure, ifenprodil blocked glutamate-induced aberrant calcium influx, suggesting the crucial role of NR2B over-expression in triggering neuronal hyper-excitability. Furthermore, ifenprodil treatment was able to prevent NR2A subunit over-expression by means of ERK1/2 phosphorylation. Our findings indicate oxidative stress and NR2B/NMDA signaling as promising therapeutic targets for co-treatments aimed to prevent chronic epilepsy following the seizure onset. http://repub.eur.nl/res/pub/34467/ 2011-08-15T00:00:00Z Aims: The study of the intracellular oxido-reductive (redox) state is of extreme relevance to the dopamine (DA) neurons of the substantia nigra pars compacta. These cells possess a distinct physiology intrinsically associated with elevated reactive oxygen species production, and they selectively degenerate in Parkinson's disease under oxidative stress conditions. To test the hypothesis that these cells display a unique redox response to mild, physiologically relevant oxidative insults when compared with other neuronal populations, we sought to develop a novel method for quantitatively assessing mild variations in intracellular redox state. Results: We have developed a new imaging strategy to study redox variations in single cells, which is sensitive enough to detect changes within the physiological range. We studied DA neurons' physiological redox response in biological systems of increasing complexity-from primary cultures to zebrafish larvae, to mammalian brains-and identified a redox response that is distinctive for substantia nigra pars compacta DA neurons. We studied simultaneously, and in the same cells, redox state and signaling activation and found that these phenomena are synchronized. Innovation: The redox histochemistry method we have developed allows for sensitive quantification of intracellular redox state in situ. As this method is compatible with traditional immunohistochemical techniques, it can be applied to diverse settings to investigate, in theory, any cell type of interest. Conclusion: Although the technique we have developed is of general interest, these findings provide insights into the biology of DA neurons in health and disease and may have implications for therapeutic intervention. http://repub.eur.nl/res/pub/34069/ 2011-06-01T00:00:00Z The molecular basis for epileptogenesis remains poorly defined, but repeated or prolonged seizures can cause altered hippocampal N-methyl d-aspartate receptor (NMDAR) stoichiometry, loss of hippocampal neurons, and aberrant mossy fiber sprouting. Using the muscarinic receptor 1 (m1R) agonist, pilocarpine (PILO), in hippocampal cell cultures we explored the early sequence of molecular events that occur within 24 h of the initial insult and result in altered neuronal function during epileptogenesis. Our findings show that PILO-induced, m1R-mediated, inositol 1,4,5-trisphosphate (IP3) synthesis constitutes an early, crucial biochemical event required for NMDAR hyperactivation and subsequent NADPH oxidase (NOX) activation and NMDAR-independent ERK1/2 phoshorylation. Together, but not separately, NOX activation and ERK1/2 phosphorylation induce alterations in NMDAR stoichiometry through the upregulation of NR1 and NR2B subunits. Lastly, we demonstrated that PILO-mediated oxidative stress alters NMDAR function through the redox modulation of cysteine residues. The in vitro results related to thiol oxidation, NOX activation, ERK1/2 phosphorylation and NMDAR upregulation were confirmed in vivo, 24 h after treatment of adult rats with PILO. These results obtained in PILO-treated primary hippocampal neurons - and confirmed in vivo at the same time-point after PILO - provide a better understanding of the early cellular responses during epileptogenesis and identify potential therapeutic targets to prevent development of chronic epilepsy.