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。它是新生儿癫痫发作的最常见原因，可导致显著的神经发育障碍。目前可用于新生儿癫痫发作的治疗方法疗效有限，动物数据表明它们可能具有显著的毒性。缺氧导致癫痫易感性增加的机制尚不完全清楚。在初步实验中，我们发现神经一氧化氮合酶（nNOS）的药物抑制可防止未成熟啮齿动物的缺氧再氧化癫痫发作。此外，我们还发现缺氧再氧化和外源性一氧化氮（NO）暴露都会导致P7小鼠CA1神经元的神经元兴奋性增加。除了膜特性的改变外，NO暴露还导致培养海马神经元中谷氨酸能和氨基丁酸能突触传递增强。最后，我们的初步数据还显示，NO增加了培养海马神经元的端内钙浓度。在本实验中，我们将验证缺氧再氧化过程中nnos介导的NO产生导致新生儿海马神经元高兴奋性和突触传递功能障碍的假设。采用野生型和nNOS敲除小鼠，我们将完成以下3个目的：(1)利用电流钳电生理学研究缺氧-再氧化和NO对CA1神经元兴奋性的影响；(2)应用电压钳电生理技术研究缺氧-复氧和NO对CA1锥体神经元谷氨酸能和gaba能突触传递的影响；(3)利用钙成像和共聚焦显微镜技术确定NO对突触钙动力学的影响。实验将在弗吉尼亚大学进行，得到儿科的支持，并在电生理学领域专家Kapur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，这将使候选人处于一个理想的位置，以研究缺氧引起的癫痫对未成熟大脑的急性和长期影响。

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.