Extracellular Potassium Regulates the Chloride Reversal Potential in Cultured Hippocampal Neurons

Brain Res. 2008 Apr 18;1205:12-20. doi: 10.1016/j.brainres.2008.02.038. Epub 2008 Feb 26.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / physiology
  • Algorithms
  • Animals
  • Blotting, Western
  • Cells, Cultured
  • Chlorides / metabolism*
  • Electrophysiology
  • Female
  • Hippocampus / cytology*
  • Hippocampus / drug effects
  • Membrane Potentials / drug effects
  • Neurons / drug effects*
  • Patch-Clamp Techniques
  • Potassium / pharmacology*
  • Pregnancy
  • Rats
  • Sodium-Potassium-Chloride Symporters / metabolism
  • Solute Carrier Family 12, Member 2
  • Symporters / metabolism
  • Synaptic Transmission / drug effects
  • gamma-Aminobutyric Acid / physiology

Substances

  • Chlorides
  • Slc12a2 protein, rat
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 2
  • Symporters
  • potassium-chloride symporters
  • gamma-Aminobutyric Acid
  • Potassium