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Keywords:

  • calcium current;
  • isolated cells;
  • patch-clamp;
  • potassium current;
  • sodium current

Abstract

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about –45 mV, was half-maximal steady-state inactivated at –66.6 mV, and recovery from inactivation followed a two-exponential process (τ1 = 8.4 ms and τ2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm) and a second by high concentrations (6 mm). After elimination of Ifast by a conditioning prepulse (50 ms to –50 mV), a slow K+ current (IKV) could be studied in isolation. IKV was only moderately affected by 4-AP (6 mm), while TEA (20 mm) blocked most (> 80%) of the current. IKV activated at about –40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at –30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and IKV, indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and IKV were blocked by tetrodotoxin (TTX, 1 μm), 4-AP (6 mm) and TEA (20 mm) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about –30 mV, were completely blocked by 50 μm cadmium (Cd2+), were sensitive to verapamil (≈ 40% block by 10 μm), and were not affected by nickel (50 μm). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).