Characterization of sodium current in developing rat diencephalic neurons in serum-free culture

Abstract

1. Dissociated, synchronized (G1 phase of cell cycle), and birth-dated fetal rat diencephalic neurons were grown in a serum-free defined medium. The gigaseal whole-cell voltage-clamp technique was used to measure the inward Na+ currents (INa) from morphologically identified bipolar neurons. The earliest expressed somatic INa has been characterized and compared with that present at a later date. 2. The identity of the INa was established on the basis of its reversal potential and reversible blockade by tetrodotoxin (TTX). Close agreement between the measured reversal potentials (68.5 +/- 1.3 and 38.3 +/- 2.4 mV, mean +/- SE) and calculated Nernst equilibrium potentials (64.6 and 34.7 mV) at two different bath Na+ concentrations (120 and 35 mM, respectively) suggests that the channels are highly selective for Na+. 3. The peak INa density increased from 47.7 +/- 2.9 pA/pF in younger neurons (5-6 days in culture) to 93.9 +/- 6.4 pA/pF in older neurons (12-13 days in culture). The activation voltage and the voltage for peak current were also shifted by 10 mV in the hyperpolarizing direction, from -30 and +10 mV in younger neurons to -40 and 0 mV in older neurons, respectively. However, the reversal potential did not change (69.2 +/- 2.3 and 68.5 +/- 1.3 mV in younger and older neurons, respectively). 4. In older neurons the steady-state inactivation parameters (V1/2, the voltage at which inactivation was 50% of maximum, and kh, the voltage at which there is an e-fold change in inactivation) were significantly altered. V1/2 was shifted from -41.5 +/- 2.3 to -48.8 +/- 1.8 mV, and kh was increased from 6.2 +/- 0.5 to 8.9 +/- 0.4 mV. However, the time course of activation and the rates of inactivation and recovery from inactivation were unchanged. 5. In both groups, the INa decays were best described by a sum of two exponentials. The corresponding time constants were voltage dependent. Also, the amplitudes of the two components were differentially affected by membrane potential and niflumic acid. 6. The extrapolated amplitudes of both the fast and the slow components of INa were larger in older neurons, but the ratio of the amplitudes of the two components did not change with age. The voltage dependencies of the time constants of both components were altered. 7. We conclude that INa in fetal rat diencephalic neurons grown in a defined medium with only essential nutrients undergoes in vitro changes in current density and in some, but not all, kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)