Extracellular potassium regulates the chloride reversal potential in cultured hippocampal neurons.
Inhibitory GABAergic synaptic transmission in the mammalian hippocampus depends upon a hyperpolarized reversal potential for Cl(-) (ECl). To examine the regulation of ECl hyperpolarization we cultured hippocampal neurons for two weeks in either a low- or a high-concentration of KCl (2.6 or 18.7 mM, respectively). Neurons were then recorded from standard extracellular solution containing 3 mM K+, using the dual perforated patch clamp technique. Low-KCl cultured neurons fired spontaneous action potentials (APs; 0.33+/-0.11 Hz), while high-KCl cultured neurons were quiescent, resulting in a significant difference in AP activity (p=0.042). This high-KCl-induced decrease in activity was accompanied by depolarizations of both the AP threshold (p<0.001) and ECl (p<0.001), and a decrease in input resistance (IR, p<0.001), when compared with low-KCl cultured neurons. Blocking AP firing of low-KCl neurons during culturing with 1 muM tetrodotoxin did not alter ECl hyperpolarization, when compared with drug-free cultured low-KCl neurons (p=0.627); thus AP firing is not required for ECl hyperpolarization. Acute perfusion of a high-KCl extracellular solution onto low- or high-KCl cultured neurons demonstrated that high-KCl significantly depolarized the resting membrane potential (RMP). The KCl-induced change in ECl did not correspond with alterations in the expression of the cation chloride cotransporters KCC2 and NKCC1, as determined by western blotting (p=0.736). These findings suggest that: (1) extracellular K+ regulates ECl hyperpolarization; and, (2) the use of high-KCl during neuronal culture produces biophysically abnormal parameters, and thus should be discouraged.
Balena
Trevor
T
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario Canada M5S 3G5.
Acton
Brooke A
BA
Koval
Dmitrii
D
Woodin
Melanie A
MA
D000200:Action Potentials / Q000502:physiology; D000465:Algorithms; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004594:Electrophysiology; D005260:Female; D006624:Hippocampus / Q000166:cytology* / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology*; D011247:Pregnancy; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects; D005680:gamma-Aminobutyric Acid / Q000502:physiology; D000096922:K Cl- Cotransporters
D002712:Chlorides; D011188:Potassium; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters; C578879:Slc12a2 protein, rat