The experimental protocols useful for all studies described here conform strictly to the guidelines of the Institutional Animal Use and Care Committee (IACUC) of CDC-NIOSH and of the CUMC

The experimental protocols useful for all studies described here conform strictly to the guidelines of the Institutional Animal Use and Care Committee (IACUC) of CDC-NIOSH and of the CUMC. Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies. Wang et al., (2005) using CNSProfile to monitor the state of phosphorylation of neuronal signaling proteins. Common patterns of protein phosphorylation changes were evident after DFP treatment of two different mouse strains (C57BL/6 and FVB) with different sensitivities to the nerve agent. Because these changes correlate with onset of CNS symptoms of nerve agent toxicity they represent important signaling targets for nerve agents that will be useful for the development of more effective treatments to block or attenuate short-term and long-term nerve agent effects. Female Necrosulfonamide C57BL/6 mice displayed dose-dependent increases in seizure-like behavior in response to DFP within 5 min after nerve agent administration, often culminating in death within 20C30 min. Male FVB mice of a similar age and body weight also developed seizure-like behaviors rapidly (within 5C10 min) after DFP injections, but exhibited sustained seizure-like symptoms for several hours with lower overall lethality. In both mouse strains, DFP exposure elicited comparable site- and region-specific effects on phosphorylation of several signaling phosphoproteins in the brain that correlated with the onset of the most severe seizure-related behaviors. Phosphorylation site changes were typically observed by 15 min in the female C57BL/6 mouse brains, whereas most phosphorylation changes in the brains of male FVB were most pronounced at 2h after nerve agent exposure. A major effect of DFP exposure in mice is the alteration of the state of phosphorylation of regulatory residues on glutamate receptors, including S897 of the Necrosulfonamide NR1 NMDA receptor subunit (Tingley et al., 1997). These data are consistent with reports that nerve agents induce a sequential activation of distinct neurochemical systems in the brain resulting in a delayed recruitment of glutamatergic neurons (Shih & McDonough; 1997; Shih et al., 2003). A rapid reduction was seen in the level of NR1 phosphorylated at the S897 residue in mouse striatum at the earliest time point monitored after DFP exposure (15 min in female C57BL/6 mice and 30 min in male FVB mice). Previous work from our laboratory (Snyder et al., 1998) has shown that the phosphorylation state of S897 on NR1 in striatum is under the control of a PKA-dependent signaling cascade that is reciprocally regulated by both dopamine and glutamate neurotransmission. Phosphorylation of NR1 S897 accentuates NMDA receptor signaling, increasing gene transcription involving CREB (Dudman et al., 2003) and reducing receptor removal from the plasma membrane (Scott et al., 2003). We interpret the profound dephosphorylation of striatal S897 NR1 observed after DFP exposure as a signal subsequent to elevated glutamatergic activity which occurs as the delayed response to the nerve agent. Dephosphorylation of this site in response to glutamate overactivity could be anticipated to dampen glutamate effects by attenuating gene expression effects via CREB, and reducing receptors in the plasma membrane. In contrast, NR1 phosphorylation in hippocampus was upregulated after DFP exposure. Rabbit Polyclonal to Paxillin (phospho-Ser178) S897 phosphorylation was elevated by 75% in hippocampus, relative to vehicle-treated control mice. The biochemical basis for the bi-directional regulation of S897 phosphorylation in these two brain regions is unclear. The increase in NR1 phosphorylation state was delayed until 2 h after Necrosulfonamide DFP treatments, compared to striatal NR1, which was significantly dephosphorylated at 30 min after DFP exposure. One plausible hypothesis for the difference in NMDA receptor phosphorylation in these two brain regions is that rapid activation of protein phosphatase-1, governed by the DARPP-32 cascade, leads to striatal dephosphorylation Necrosulfonamide of NR1. Since DARPP-32 is enriched in striatal medium spiny-type neurons, but is essentially undetectable in hippocampus, this would explain the distinct hippocampal pattern (Ouimet et al., 1984) Future studies comparing NR1 phosphorylation in response to DFP in wildtype versus DARPP-32 knockout mice will be useful in testing this hypothesis. In addition to the effects of NR1 phosphorylation, increased phosphorylation of GluR1 residues is believed to enhance AMPA receptor activity (Derkach et.