Endogenous acidification of the inhibitory synapse : proton amplification of GABAA-mediated neurotransmission
Dietrich, Craig Julius.
Thesis (Ph.D.)--Georgetown University, 2009.; Includes bibliographical references. Maintenance of external pH is critical to ensuring proper CNS function. Recent work in excitatory transmission suggests that in vivo synaptic proton buffering is not sufficient to rigidly maintain an extracellular pH of 7.4. To extend this work to inhibitory synapses, I recorded miniature GABA inhibitory post-synaptic currents (mIPSCs) from cultured rat cerebellar granule cells (CGCs) under varying pH and proton buffering conditions. Consistent with previous findings in rat hippocampal neurons, mIPSCs recorded from CGCs under rigid extracellular pH control (achieved by using 24 mM HEPES) exhibited an inverse relationship between extracellular pH and event size, such that raising pH from 6.8 to 8.0 resulted in a nearly 50% decrease in charge transfer. Kinetically, alkalinization sped both the mIPSC rise-time and fast component of decay while slowing the slow component. More intriguingly, I found that while maintaining external pH at 7.4, increasing the proton buffering capacity of the extracellular solution from 3 mM to 24 mM HEPES mimicked changes in amplitude and kinetics consistent with alkalinization. Supplementing physiological buffer concentrations (24 mM HCO3-/5% CO2) with 10 mM HEPES also reduced mIPSCs in a manner consistent with alkalinization, providing further support of an endogenous acidifying force at the synapse. In this study, I found evidence that the Na+/H+ ion exchanger (NHE) is the source of this synaptic acidification. Inhibition of the exchanger with either amiloride (20 μM) or replacement of extracellular sodium with lithium also resulted in mIPSC inhibition consistent with alkalinization. The effects of increased proton buffering via HEPES and NHE inhibition via amiloride were negated by acidification to pH 6.8. Furthermore, the effects of NHE inhibition on mIPSCs was confirmed in 24 mM HCO3-/5% CO2 buffering. Furthermore, I found that NHE inhibition with 1mM lithium in physiological buffering conditions was sufficient to cause significant reduction in mIPSCs. These results provide strong evidence that endogenous acidification of the GABAergic synapse via the Na+/H+ exchanger is of sufficient magnitude to enhance inhibitory neurotransmission.
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Queenan, Bridget Noelle (Georgetown University, 2014)The central nervous system (CNS) is a living, breathing electrical circuit which learns and remembers by altering its own connectivity. Neurons, the electrical cells of the CNS, are thought to encode information by adjusting ...