Effects of Nitric Oxide on Synaptic Transmission in the Neonatal Hippocampus

描述(申请人提供)：缺氧缺血性脑损伤是围产期医学的主要问题。它是新生儿癫痫最常见的原因，可导致严重的神经发育障碍。目前对新生儿癫痫发作的治疗效果有限，动物数据表明它们可能有显著的毒性。低氧导致癫痫敏感性增加的机制尚不完全清楚。在初步实验中，我们发现对神经元型一氧化氮合酶(NNOS)的药理抑制可以防止幼年啮齿动物发生缺氧-复氧发作。此外，我们还发现，低氧-复氧和外源性一氧化氮(NO)暴露均导致P7小鼠CA1神经元兴奋性增加。除了细胞膜特性的改变外，NO暴露还导致培养的海马神经元谷氨酸和GABA能突触传递增强。最后，我们的初步数据还表明，NO增加了培养的海马神经元的端粒内钙离子浓度。在拟议的实验中，我们将检验nNOS介导的NO在缺氧-复氧过程中导致新生海马神经元过度兴奋和突触传递功能障碍的假设。利用野生型和nNOS基因敲除小鼠，我们将完成以下三个目标：(1)利用电流钳电生理学研究缺氧-复氧和NO对CA1神经元兴奋性的影响；(2)利用电压钳电生理技术研究缺氧-复氧和NO对CA1锥体神经元谷氨酸和GABA能突触传递的影响；(3)利用钙成像和共聚焦显微镜技术观察NO对突触钙动力学的影响。实验将在弗吉尼亚大学儿科系的支持下进行，并在电生理领域的专家卡普尔博士的指导下进行。在Kapur博士的指导下，候选人将获得新的技能，包括膜片钳电生理学和体外突触钙成像，这将使候选人处于研究缺氧诱导的未成熟大脑癫痫的急性和长期影响的理想位置。 与公共卫生相关：识别缺氧高兴奋性的机制，如NO介导的机制，可能会为开发治疗新生儿癫痫的新疗法开辟新的途径。这是一个重要的研究领域，因为目前对癫痫新生儿可用的治疗方法的疗效有限、可能出现急性毒性和长期不良影响。

DESCRIPTION (provided by applicant): Hypoxic-ischemic brain injury is a major problem in perinatal medicine. It is the most common cause of seizures in the newborn and can lead to significant neurodevelopmental impairments. Current therapies available to neonates with seizures have limited efficacy and animal data suggest they may have significant toxicities. The mechanisms by which hypoxia leads to increased seizure susceptibility are incompletely understood. In preliminary experiments, we found that pharmacologic inhibition of neuronal nitric oxide synthase (nNOS) prevents hypoxia-reoxygenation seizures in immature rodents. Further, we also found that both hypoxia-reoxygenation and exogenous nitric oxide (NO) exposure resulted in increased neuronal excitability in CA1 neurons from P7 mice. In addition to changes in membrane properties, NO exposure resulted in enhanced glutamatergic and GABAergic synaptic transmission in cultured hippocampal neurons. Finally, our preliminary data also shows that NO increases intraterminal calcium concentration in cultured hippocampal neurons. In the proposed experiments, we will test the hypothesis that nNOS-mediated NO production during hypoxia-reoxygenation leads to neuronal hyperexcitability and synaptic transmission dysfunction in the neonatal hippocampus. Using wild-type and nNOS knockout mice , we will complete the following 3 aims: (1) To investigate the effects of hypoxia-reoxygenation and NO on CA1 neuronal excitability using current-clamp electrophysiology; (2) To investigate the effects of hypoxia-reoxygenation and NO on glutamatergic and GABAergic synaptic transmission in CA1 pyramidal neurons using voltage-clamp electrophysiology; and (3) To determine the effects of NO on synaptic calcium dynamics using using calcium imaging and confocal microscopy techniques. Experiments will be conducted at the University of Virginia with the support of the Department of Pediatrics and under the mentorship of Dr Kapur, an expert in the field of electrophysiology. With Dr Kapur's guidance the candidate will acquire new skills, including patch-clamp electrophysiology and in vitro synaptic calcium imaging, that will place the candidate in an ideal position to study the acute and long term effects of hypoxia-induced seizures in the immature brain. PUBLIC HEALTH RELEVANCE: The identification of the mechanisms of hypoxic hyperexcitability such as those mediated by NO may lead to novel pathways for the development of new therapies for neonatal seizures. This is an important area of research because of the limited efficacy, possible acute toxicities and long-term adverse effects of the treatments currently available to newborn with seizures.