BRAIN GALANIN RECEPTORS AND EPILEPSY

描述(申请人提供)：本项目的长期目标是了解神经肽甘丙肽抑制癫痫发作活动的机制。我们的实验室首次证明癫痫发作会改变海马体中的甘丙素，而且甘丙素在癫痫持续状态中具有深刻的癫痫保护作用。该项目有三个目标。首先，我们将开发一种新的方法来下调幼年动物的甘丙素受体，以研究甘丙素受体在癫痫发作中的作用。这将通过开发和优化针对甘丙肽受体亚型1和2的反义多肽核酸(PNA)来实现。通过研究甘丙肽受体结合、免疫细胞化学和Western印迹，我们将在甘丙肽受体转基因细胞系和原代神经元培养中检测PNA对甘丙肽受体的影响。其次，我们将验证大脑中甘丙肽受体亚型下调选择性地增加动物癫痫易感性，并增加癫痫相关神经元损伤的假设。我们将检验幼稚和甘丙肽受体PNA处理的大鼠在自持性癫痫持续状态期间癫痫发作活动的启动和维持的差异，并将比较对照组和PNA处理的大鼠的神经元损伤程度。第三，我们将研究甘丙肽受体刺激的癫痫保护作用的可能机制。具体地说，我们将研究甘丙肽受体PNA治疗如何影响第二信使系统(环磷酸腺苷、磷脂酰肌醇周转率、丝裂原激活蛋白激酶活性、兴奋性氨基酸谷氨酸释放)，并将这些影响与PNA对癫痫持续状态的影响相关联。这项拟议的项目将为理解参与内源性抑制癫痫的大脑机制提供重要数据，并将证明使用甘丙肽受体的活性配体作为治疗癫痫的新方法是合理的。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the mechanisms involved in inhibition of seizure activity by the neuropeptide galanin. Our lab was the first to show that seizures alter galanin in the hippocampus and that galanin has a profound seizure-protective effect in status epilepticus. The project has three goals. First, we will develop a novel approach to down-regulate galanin receptors in naive animals, in order to study the role of galanin receptors in seizures. This will be achieved by developing and optimizing peptide nucleic acid (PNA) antisense targeted against galanin receptor subtypes 1 and 2. The effects of PNA on galanin receptors will be examined by studying galanin receptor binding, immunocytochemistry, and Western blot in galanin receptor-transfected cell lines and primary neuronal cultures. Second we will test the hypothesis that down-regulation of galanin receptor subtypes in the brain selectively increases animals' predisposition to seizures, and increases seizure-related neuronal injury. We will examine the differences between naive and galanin receptor PNA-treated rats in the initiation and maintenance of seizure activity during self-sustaining status epilepticus, and will compare the degree of neuronal injury between control and PNA-treated rats. Third, we will study putative mechanisms that underlie seizure-protective effects of galanin receptor stimulation. Specifically, we will study how galanin receptor PNA treatment affects second messenger systems (cyclic adenosine monophosphate, phosphoinisitide turnover, mitogen-activated protein kinase activity, excitatory amino acid glutamate release) and will correlate these effects with the effects of PNA on status epilepicus. The proposed project will yield important data for understanding brain mechanisms that participate in endogenous suppression of seizures, and will justify the use of active ligands of galanin receptors as a new approach for the treatment of epilepsy.