PROJECT SUMMARY/ABSTRACT There are no pharmacological treatments for cytotoxic edema, which is a common consequence of multiple brain insults, including hypoxic-ischemic and traumatic brain injury, stroke, metabolic derangements, and seizures. Hypoxic-ischemic encephalopathy (HIE), with an incidence of 1.5 of every 1,000 live births, is a type of brain damage in newborns caused by oxygen deprivation and limited blood flow. HIE is associated with seizures, and both correlate with long-term morbidity, including cerebral palsy, cognitive delay, epilepsy, vision loss, and deafness. HIE and neonatal seizures result in cytotoxic edema, which is characterized by the accumulation of water, chloride (Cl-), and other ions. The mechanisms of water movement that make neurons swell during the neonatal period are unknown. There is a critical need to determine how water moves into neurons that result and perpetuate neuronal swelling during the neonatal period, as there are no direct treatments for cytotoxic edema at this age. Knowing the pathways of water movement in neurons is the first step to develop innovative ways to treat cytotoxic edema, which will prevent neuronal cell death and improve the treatment of neonatal seizures. Neurons do not have water channels to allow the movement of water. Multiple pathways have been described in different cell types, but it is unknown which ones participate during the neonatal period. Our long- term goal is to identify the mechanisms of neuronal swelling in the developing brain and how this swelling results in neuronal death. Our central hypothesis for this proposal is that specific cation-chloride cotransporters (CCCs) move water, along with Cl-, in and out of neurons during cytotoxic edema in the neonatal period. This hypothesis is based on our data demonstrating the linked movement of water and Cl- in neurons during cytotoxic edema. We will test our hypothesis through two specific aims. Aim 1 will determine the pathway of water movement into neurons during swelling in the neonatal period. Aim 2 will determine the paths of water movement out of cortical neurons that prevent progressive swelling during the neonatal period. We will use multiphoton imaging techniques to measure changes in neuronal size and their Cl- concentration during swelling in different transgenic mouse lines expressing both Cl- sensitive and insensitive fluorophores, in vitro, and in vivo, while altering the CCC function either pharmacologically or through genetic manipulation. Also, we will use a novel deep learning algorithm to analyze the changes in neuronal size during swelling. Our studies will uncover fundamental mechanisms on how neurons swell and what mechanisms prevent progressive swelling during early brain development. Our results will have a broad impact as they will open new research avenues on neuronal volume regulation in the newborn and will guide the development of drugs targeting cytotoxic edema, which are currently lacking. Moreov...