Mechanism and Treatment of nerve agent-induced seizures

描述(由申请人提供)：这项提案建议开发新的、基于机制的治疗方法，用于治疗有机磷(OP)神经毒剂诱导的癫痫。OP神经毒剂，如沙林、梭曼和VX，通过抑制中枢和外周胆碱酯酶发挥作用，增强胆碱能传递。在实验动物中，所有已知的神经毒剂在接触后几分钟内会导致惊厥发作和癫痫持续状态。在两伊战争期间暴露于神经毒剂中毒的人类，以及使用沙林和VX的东京地铁袭击中，也观察到了癫痫发作。OP诱发癫痫的机制尚不清楚。我们实验室的初步研究已经使用OP，对氧磷作为替代药物来研究OP诱导的癫痫发作。我们证明，低剂量的对氧磷注入海马体会导致长时间的癫痫发作。此外，我们证明了在海马片中引起癫痫发作的对氧磷浓度可以通过刺激突触前终末释放谷氨酸来增强兴奋性神经传递。这些发现导致了指导这一提议的中心假说的形成：惊厥浓度的OP神经毒剂增强了谷氨酸能神经传递。这项建议旨在证实OP制剂对兴奋性传递的影响的初步发现，并测试减少突触前终末谷氨酸释放的候选化合物作为对抗胆碱能药物诱导的癫痫的潜在治疗方法。实验是在三个目标下提出的，每组都有针对治疗干预的具体里程碑。我们提出了代表“去不去”决策点的量化结果标准。目的1利用膜片钳电生理学和FM染色技术，试图证实和扩大OP胆碱酯酶抑制剂促进海马区突触前终末谷氨酸释放的观察结果。目标2中提出的实验试图测试两类化合物，生长抑素及其类似物，腺苷及其类似物，在控制休息条件下和刺激OP后减少谷氨酸释放的能力，使用膜片钳技术和FM染色技术。甘丙素和神经肽Y两类化合物被保留下来，将在两种拟议药物中的任何一种未能将谷氨酸释放减少30%的情况下进行测试。目的：检测两类化合物对锂/匹罗卡品和对氧磷致癫痫持续状态的抑制作用。神经肽Y和甘丙素两类化合物被保留下来，将在两种拟议药物中的任何一种未能控制50%的实验动物的安定难治性癫痫发作的情况下进行测试。通过研究OP诱发癫痫的机制，我们建议为OP诱发癫痫的治疗寻找新的治疗靶点。其次，我们建议开发基于新的治疗靶点的治疗方法。此外，我们还开发了一种可用于民用的OP诱发癫痫模型

DESCRIPTION (provided by applicant): This proposal proposes to develop novel, mechanism-based therapies for the treatment of organophosphate (OP) nerve agent-induced seizures. OP nerve agents, such as sarin, soman and VX act by central and peripheral cholinesterase inhibition and enhance cholinergic transmission. In experimental animals, all known nerve agents, produce convulsive seizures and status epilepticus within minutes of exposure. Seizures were also observed in humans exposed to nerve agent poisoning during the Iran-Iraq war, and in the Tokyo subway attacks where sarin and VX were used. The mechanism of OP-induced seizures remains uncertain. Preliminary studies in our laboratory have used the OP, paraoxon, as a surrogate agent to study OP-induced seizures. We demonstrate that low doses of paraoxon infused into the hippocampus cause prolonged seizures. Furthermore, we demonstrate that the concentrations of paraoxon that cause seizures in hippocampal slices can enhance excitatory neurotransmission by stimulating glutamate release from presynaptic terminals. These findings lead to the formulation of the central hypothesis guiding this proposal: convulsant concentrations of OP nerve agents enhance glutamatergic neurotransmission. This proposal seeks to confirm preliminary findings regarding the effect of OP agent on excitatory transmission and to test candidate compounds that diminish glutamate release from presynaptic terminals as potential therapy against seizures induced by cholinergic agents. Experiments are proposed within three aims, each group has specific milestones toward therapeutic interventions. We present quantitative outcomes criteria that represent "go-no go" decision points. Experiments outlined in Aim 1 seek to confirm and extend the observation that OP cholinesterase inhibitors enhance glutamate release from presynaptic terminals in the hippocampus, using patch clamp electrophysiology and FM dye technique. Experiments proposed in Aim 2 seek to test two classes of compounds, somatostatin and its analogs, and adenosine and its analogs, for their ability to diminish glutamate release during control resting conditions and following OP stimulation, using patch clamp technique and FM dye technique. Two classes of compounds, galanin and neuropeptide Y, are held in reserve and will be tested in case either one of the two proposed agents fail to reduce glutamate release by 30%. Aim 3: To test anticonvulsant action of two classes of compounds in controlling lithium/pilocarpine and OP paraoxon-induced status epilepticus. Two classes of compounds, neuropeptide Y and galanin are held in reserve and will be tested in case either one of the two proposed agents fail to control diazepam refractory seizures in 50% of experimental animals. By studying the mechanisms of OP-induced seizures, we are proposing to identify novel therapeutic targets for the treatment of OP-induced seizures. Second, we propose to develop therapies based on novel therapeutic targets. Furthermore, we develop a model of OP-induced seizures that can be used in civilian