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.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Clinical Investigator Award (CIA) (K08)
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Neurological Sciences Training Initial Review Group (NST)
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Whittemore, Vicky R
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University of Virginia
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Zanelli, S A; Rajasekaran, K; Grosenbaugh, D K et al. (2015) Increased excitability and excitatory synaptic transmission during in vitro ischemia in the neonatal mouse hippocampus. Neuroscience 310:279-89
Zanelli, S; Goodkin, H P; Kowalski, S et al. (2014) Impact of transient acute hypoxia on the developing mouse EEG. Neurobiol Dis 68:37-46
Rajasekaran, Karthik; Zanelli, Santina A; Goodkin, Howard P (2010) Lessons from the laboratory: the pathophysiology, and consequences of status epilepticus. Semin Pediatr Neurol 17:136-43