GABA Induced Depolarization: A Tale of Opposing Forces December 2010 Jong M. Rho, MD Alberta Children s Hospital University of Calgary, Alberta, Canada American Epilepsy Society Annual Meeting
Disclosures National Institutes of Health Arizona Biomedical Research Commission Barrow Neurological Foundation Alberta Children s Hospital Foundation & Research Institute Ortho McNeil Neurologics Questcor Pharmaceuticals Xenome Pharmaceuticals SHS/Nutricia Sunovion Pharmaceuticals Lundbeck Pharmaceuticals Eisai Pharmaceuticals Research Grant Funding Research Grant Funding Research Grant Funding Research Grant Funding Research Grant Funding Research Grant Funding Research Grant Funding Research Grant Funding Consultant Speaker s Bureau Speaker s Bureau American Epilepsy Society Annual Meeting
The Central Inhibitory Synapse Astrocyte GABAergic neuron Cholesterol Pregnenolone Neurosteroids Neurosteroids GABA receptors A Localization Synaptic Extrasynaptic Positive modulators Function Phasic inhibition Tonic inhibition Benzodizepines Barbiturates Felbamate Topiramate Neurosteroids, alcohol, general anesthetics Modified from Benarroch. Neurology 2007
Molecular Structure & Composition of GABA A Receptors N C GABA α1 α6 Cl β1 β3 γ 1 γ 3 or δ, ε, π, θ M1 M2 M3 M4 5 Inhibitory postsynaptic potential Benarroch. Neurology 2007
Mechanism of GABA Induced Depolarization Cl K + Cl GABA receptor Na + K + 2Cl Cl NKCC1 Cl KCC2 Depolarization Hyperpolarization GABA GABA Kahle et al. Nat Clin Pract Neurol 2008
Adult = inhibition = excitation Immature Modified from Rivera et al, J Physiol 2005
What is the Laboratory Evidence for GABA Depolarization in Developing Brain? Abundant electrophysiological evidence in multiple regions of the brain Expression studies of cation chloride cotransporters (i.e., NKCC1 and KCC2) that are consistent with GABA depolarization Consistent effects of the NKCC1 blocker bumetanide under various experimental conditions
Efficacy of GABAergic Anticonvulsants in Animal Models of Neonatal Seizures Dzhala et al, J Neurosci 2010
Effects of NKCC1 Inhibition on E GABA And Spontaneous Network Activity Dzhala et al, Nat Med 2005
Bumetanide Enhances Phenobarbital Efficacy in a Neonatal (P5) Seizure Model Dzhala et al, Ann Neurol 2008
What is the Clinical Evidence for GABA Depolarization in Developing Brain? GABAergic drugs not consistently effective in newborns and young infants As a corollary, efficacy seen may be due to mechanisms other than GABA A receptor modulation GABA induced depolarization, and differential patterns of cation chloride cotransporter expression in surgicallyresected epileptic tissues
NEJM 1999
Evidence from Surgically Resected Human Epileptic Tissues Malformations of cortical development Aronica et al, Neurosci 2007 Munakata et al, Epilepsia 2007 Temporal lobe epilepsy Palma et al, Proc Natl Acad Sci 2006 Huberfeld et al, J Neurosci 2007 Hypothalamic hamartoma Kim et al, Epilepsia 2008 Wu et al, Exp Neurol 2009
Decreased Seizure Activity in a Human Neonate Treated with Bumetanide BMT, bumetanide; 0.1 mg/kg Kahle et al, J Child Neurol 2009
A Metabolic Twist
Brain Metabolism & Development Prins, J Cereb Blood Flow Metab 2008
Effects of Ketones on the Immature Brain Prins, J Cereb Blood Flow Metab 2008
β Hydroxybutyrate Shifts GABA Reversal Potentials Rheims et al, J Neurochem 2009
β Hydroxybutyrate Shifts GABA Reversal Potentials Rheims et al, J Neurochem 2009
Metabolic Substrates Alter GABAergic Signaling Holmgren et al, J Neurochem 2010
Hypothetical Scheme Summarizing Effects of Energy Substrates on Neuronal RMP and [Cl] i Holmgren et al, J Neurochem 2010
Incongruency of Results
GABA Depolarizes Immature Neocortical Neurons in the Presence of the Ketone Body β Hydroxybutyrate Kirmse et al, J Neurosci 2010
GABA Depolarizes Immature Neocortical Neurons in the Presence of the Ketone Body β Hydroxybutyrate Kirmse et al, J Neurosci 2010
Depolarizing Actions of GABA in Immature Neurons Depend Neither on Ketone Bodies Nor on Pyruvate Tyzio et al, J Neurosci, in press.
Activation of GABA A Receptors Suppresses Epileptiform Activity in Neocortex of Immature Mice P5 P4 Richter et al, Epilepsia 2010
Reconciling Differences Artifact of in vitro slice preparation and/or other technical approaches? What concentrations of metabolic substrates are actually seen synaptically? Role of voltage gated chloride channels & Cl /HCO 3 exchanger? Does membrane depolarization always lead to excitation? Fluctuating metabolic state under presumed stable recording conditions?
AED Mechanisms of Action Bumetanide NKCC1
Summary GABA induced membrane depolarization and chloride homeostasis are regulated in highly complex ways especially in the developing brain The question remains: is GABA depolarization relevant in vivo? Most likely, the ontogeny/activity of cation chloride co transporters contributes only in part to seizure propensity of the immature brain Drugs like bumetanide may play a contributory role in blocking neonatal seizures