For miniature EPSC (mEPSC) recordings, neurons were placed in medium containing control solution with or without 50 m bicuculline (Tocris Bioscience) for 15 min and then returned to control medium for 30 min. protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is definitely advertised by both JNK and p38. NMDAR-dependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, induced by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is definitely induced by submembranous Ca2+, and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK YIL 781 inhibitors to ameliorate severe excitotoxic neuronal loss and ischemic cortical damage causes common apoptosis and enhances trauma-induced injury in developing neurons (Gould et al., 1994; Ikonomidou et al., 1999; Adams et al., 2004). In the adult CNS, NMDAR blockade exacerbates neuronal loss when applied after traumatic mind injury and during ongoing neurodegeneration (Ikonomidou et al., 2000), and prevents the survival of newborn neurons in the adult dentate gyrus (Tashiro et al., 2006). Molecular mechanisms of synaptic NMDAR-dependent neuroprotection are beginning to become elucidated (Hetman and Kharebava, 2006; Papadia and Hardingham, 2007; Zhang et al., 2007; Papadia et al., 2008). Therefore, reactions of neurons to NMDAR activity follow a bell-shaped curve: both too much and too little are potentially harmful. The central part of the NMDAR in CNS physiology offers an explanation as to why medical treatment of stroke with NMDAR antagonists offers failed because of poor tolerance and efficacy (Ikonomidou and Turski, 2002; Muir, 2006). It would be desirable to block pro-death NMDAR signaling in stroke, without influencing prosurvival signaling or indeed synaptic plasticity, many forms of which are mediated by NMDAR activation. In neurons, Ca2+ influx through NMDARs promotes cell death more efficiently than through additional Ca2+ channels (Tymianski et al., 1993; Arundine and Tymianski, 2004), suggesting that proteins responsible for Ca2+-dependent excitotoxicity reside within the NMDAR signaling complex (NSC). A role for the NSC in mediating NMDAR-dependent death was shown in the case of the PDZ proteins neuronal nitric oxide synthase (nNOS) and PSD-95 (Aarts et al., 2002). PSD-95 is definitely linked to the C-terminal PDZ ligand of NR2, and also binds to nNOS. When the connection of NR2B and PSD-95 is definitely disrupted, the NMDAR becomes uncoupled from nNOS activation, reducing (but not removing) NMDAR-dependent excitotoxicity (Aarts et al., 2002). The important part of nNOS and PSD-95 above some other PDZ proteins in mediating NMDAR-dependent excitotoxicity was recently shown (Cui et al., 2007). The appeal of the NSC like a restorative target YIL 781 is definitely questioned by two issues. First, it is not obvious whether NSC parts can be disrupted without influencing prosurvival or plasticity signaling. Second, NMDAR-dependent cell death can be reconstituted in non-neuronal cells lacking the NSC simply by expressing NMDARs (Cik et al., 1993; Anegawa et al., 2000), which shows that certain NMDAR-induced death pathways do not require the NSC. We have investigated both these issues with the aim of developing an effective anti-excitotoxic strategy that spares prosurvival and plasticity signaling, focusing on two important mediators of excitotoxicity: the stress-activated protein kinases (SAPKs) p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK) (Kawasaki et al., 1997; Borsello et al., 2003; Rivera-Cervantes et al., 2004). We find that these death pathways have differing requirements for the NSC and may become disrupted to great effect and without impacting prosurvival signaling or a model of NMDAR-dependent synaptic plasticity. Materials and Methods Neuronal cultures, stimulations, and assessment of nuclear morphology. Cortical neurons from embryonic day time 21 Sprague Dawley rats were cultured as explained previously (Bading and Greenberg, 1991) except that growth ZBTB16 medium was comprised of Neurobasal A medium with B27 (Invitrogen), 1% rat serum, and 1 mm glutamine. Experiments were performed after a tradition period of 9C10 d, during which neurons developed a rich network of processes, expressed practical NMDA-type and YIL 781 AMPA/kainate-type glutamate receptors, and created synaptic contacts (Hardingham et al., 2001a, 2002). Before stimulations, neurons were transferred from growth medium to a medium comprising 10% MEM (Invitrogen), 90% salt-glucose-glycine (SGG) medium (Bading et al., 1993) (114 mm NaCl, 0.219% NaHCO3, 5.292 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, 1 mm glycine, 30 mm glucose, 0.5 mm sodium pyruvate, and 0.1% phenol red; osmolarity, 325 mOsm/L). Neurons were treated with NMDA in the presence or absence of tetrodotoxin (TTX; 1 m; EMD Biosciences). To result in NMDAR-dependent excitotoxic cell death, neurons were exposed to NMDA or glutamate [in the presence of nifedipine (5 m; Tocris Bioscience)] in our standard trophically deprived medium (90% SGG and 10% MEM) for 1 h, after which neurons were washed once and returned to fresh medium. Neurons.
For miniature EPSC (mEPSC) recordings, neurons were placed in medium containing control solution with or without 50 m bicuculline (Tocris Bioscience) for 15 min and then returned to control medium for 30 min
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