The goal of this Mentored Clinical Scientist Research Career Developmental Award (K08) is to: A) Obtain skills in: in vivo multiphoton imaging, experience working with and culturing organotypic brain slices, and recording EEG in rodents. B) Have publications and preliminary data to submit a strong R01 application by the 4th year of the award. My final goal is to be an independent researcher in inhibition physiology and pediatric epilepsy, specifically in relation to traumatic brain injury (TBI). TBI is a leading caus of morbidity and mortality in the pediatric age group causing more than 50% of childhood deaths. Seizures are a common complication in pediatric TBI. How TBI leads to seizures is unclear. Several pathological conditions, including brain injury, induce an elevation of the neuronal chloride concentration [Cl-] i. The [Cl-] i am of great importance in neurons as Cl- flux through GABAA receptors mediate inhibition when [Cl-] i is lower than the neuron's resting membrane potential, but can lead to excitation if [Cl-] i is above the resting membrane potential. Our most recent publication has shown that mannitol, and osmotic agent used to treat brain edema in the Neurological Intensive Care Unit, not only decreases the neuronal volume, but also reduces [Cl-]i. Therefore, mannitol should have anti-convulsive properties. My proposal aims to answer the following hypothesis: mannitol (a prototypical hyperosmolar agent) reduces epileptiform activity in the neocortex due to a decrease in [Cl-] i. This decrease in [Cl-] i lead to an increase efficac of GABAAR mediated inhibition in a model of pediatric TBI. To answer this hypothesis, we will use in vitro / in vivo multiphoton imaging and electrophysiological techniques in a transgenic mouse that expresses a new Cl- sensitive radiometric fluorophore (SuperClomeleon). The K08 application, through courses and hands on experience, has been targeted to obtain the necessary skills in: A) multiphoton in vivo imaging; B) experience working with and culturing organotypic brain slices; C) recording and analyzing EEG in rodents. These techniques will allow me to complete my proposed project and give me the required technical knowledge to complement my current in vitro electrophysiology skills. I have a strong background in basic research and clinical pediatric neurology allowing me to understand and see the despair that families go through due to the limitations of post-traumatic epilepsy treatment. Furthermore, my 11 original published articles and my track record of competitive funding with at least one paper delivered per funding agency are evidence to my high motivation, ability to complete projects and my desire to continue a career in research targeted to neuronal inhibitory physiology and epilepsy. I am a full time faculty at Massachusetts General Hospital (MGH) in the Pediatric Neurology division of the Department of Neurology and an Instructor in Neurology at Harvard Medical School. I have the support of the Chair of the Department of Neurology and of the Chief of Pediatric Neurology to become a productive and independent researcher at MGH. With cutting edge technologies and facilities at MGH (2-photon microscopes, electrophysiological equipment), mentors with excellent track records (Dr. Staley, Dr. Bacskai), an advisory committee to discuss my research progress and translational possibilities (Dr. Rosand, Dr.
Duh aim e, Dr. Cash), the collaborative atmosphere at MGH and being part of Harvard Medical School, I am in a an excellent environment to develop a strong, productive and rewarding career as an independent researcher. The K08 training will be fundamental to help me succeed as an independent investigator.

Public Health Relevance

Seizures are a common medical problem after pediatric traumatic brain injury and it is not clear what causes it. The current proposal will test if decreasing the chloride concentration inside neurons after a brain injury model can improve seizure treatment. To answer this question, we will use microcopy imaging technologies, a newly developed fluorescent protein sensitive to chloride concentrations and neuronal electrical activity measurements.

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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Bellgowan, Patrick S F
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Massachusetts General Hospital
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Glykys, Joseph; Dzhala, Volodymyr; Egawa, Kiyoshi et al. (2017) Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential. Trends Neurosci 40:276-294
Glykys, Joseph; Staley, Kevin J (2016) Developmental Decrease of Neuronal Chloride Concentration Is Independent of Trauma in Thalamocortical Brain Slices. PLoS One 11:e0158012