During the last cycle of this grant, we established that block of inwardly-directed cation-chloride co-transport alters the neuronal steady-state chloride concentration (Cl-i) in developing neurons, and consequently improves the efficacy of GABA-mediated synaptic inhibition and the control of seizures in the newborn. This finding forms the basis of new clinical trials in the US and Europe. We also found that cation-Cl transporters are at equilibrium at resting Cl-i. However, we don't know how to reconcile this finding with data from experiments utilizing the Cl-sensitive fluorescent dual wavelength fluorophore, Clomeleon, which demonstrate that each neuron has a unique Cl-i that is quite different from its neighbors. The question we address here is: how can cation-Cl transporters be at equilibrium at so many different Cl-i? The classic view that equilibrium depends only on Cl and cation concentrations does not explain the variance in Cl-i. Neuronal cation-chloride transporters obligately move water with cations and Cl, so that these transporters move isotonic (135 mM) cation-Cl solution into and out of neurons. Thus neuronal cation-Cl transporters also transport cytoplasmic volume, which alters the cytoplasmic hydrostatic pressure. This predicts that transmembrane hydrostatic and osmotic pressure gradients contribute to the free energy of cation-Cl transport and thus the equilibrium Cl-i. For example, a neuron with lots of osmotically active protein should have a lower equilibrium Cl-i than a neuron with less protein. Our primary hypothesis is that the pressure gradient across the neuronal membrane contributes to the free energy of transport and thus the Cl-i at which transport is at equilibrium. This hypothesis has important implications for prolonged seizures, which induce changes in the neuronal cytoskeleton that increase the volume of neurons, thereby lowering the osmotic pressure. Thus a linked secondary hypothesis is that seizure-induced changes in osmotic pressure favor movement of cations, Cl-i and water into neurons via cotransporters so that Cl-i increases and GABA signaling becomes excitatory. We will test these hypotheses by measuring neuronal volume and Cl-i at steady state and in response to ionic and osmotic challenges, seizures, and specific transport inhibitors. We will use mice that genetically express Clomeleon, acute &organotypic slice preparations, in vitro and in vivo multiphoton microscopy, pH-sensitive dyes, electrophysiological recordings, and transporter phosphorylation studies. Sensitivity of cation-Cl transport to local pressure gradients would allow neurons to maintain the incredibly precise geometries needed for stable cable properties and connectivity despite dynamic subcellular and intercellular fluctuations in osmotically active protein content. The hypotheses also predict that clinically available diuretics and inhibitors of cytoskeletal changes might also be useful in the treatment of status epilepticus in both developing and mature nervous systems.

Public Health Relevance

Prolonged epileptic seizures damage the cytoskeleton of cortical neurons, allowing the neurons to expand and accumulate the salt and water needed to fill the new intracellular space. One component of salt is chloride, and its accumulation causes the neurotransmitter GABA to promote rather than inhibit seizures. We will investigate the mechanisms of chloride accumulation and strategies to prevent it in order to improve the treatment of prolonged seizures.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Clinical Neuroplasticity and Neurotransmitters Study Section (CNNT)
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Fureman, Brandy E
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Massachusetts General Hospital
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Blauwblomme, Thomas; Dzhala, Volodymyr; Staley, Kevin (2018) Transient ischemia facilitates neuronal chloride accumulation and severity of seizures. Ann Clin Transl Neurol 5:1048-1061
Costine-Bartell, Beth A; McGuone, Declan; Price, George et al. (2018) Development of a Model of Hemispheric Hypodensity (""Big Black Brain""). J Neurotrauma :
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
Glykys, Joseph; Staley, Kevin J (2015) Diazepam effect during early neonatal development correlates with neuronal Cl(.). Ann Clin Transl Neurol 2:1055-70
Staley, Kevin (2015) Molecular mechanisms of epilepsy. Nat Neurosci 18:367-72
Dzhala, Volodymyr; Staley, Kevin J (2015) Acute and chronic efficacy of bumetanide in an in vitro model of posttraumatic epileptogenesis. CNS Neurosci Ther 21:173-80
Glykys, J; Dzhala, V; Egawa, K et al. (2014) Response to comments on ""Local impermeant anions establish the neuronal chloride concentration"". Science 345:1130
Delpire, Eric; Staley, Kevin J (2014) Novel determinants of the neuronal Cl(-) concentration. J Physiol 592:4099-114
Glykys, J; Dzhala, V; Egawa, K et al. (2014) Local impermeant anions establish the neuronal chloride concentration. Science 343:670-5

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