Ca(2+) channel currents of cortical neurons from pure and mixed cultures

Abstract

Voltage-gated Ca(2+) channels (VGCCs) are key regulators of many neuronal functions, and involved in multiple central nervous system diseases. In the last 30 years, a large number of injury and disease models have been established based on cultured neurons. Culture with serum develops a mixture of neurons and glial cells, while culture without serum develops pure neurons. Both of these neuronal-culture methods are widely used. However, the properties of Ca(2+) currents in neurons from these two cultures have not been compared. In this study, we cultured rat cortical neurons in serum-containing or -free medium and then recorded the Ca(2+) channel currents using patch-clamp technique. Our results showed that there were significant differences in the amplitude and activation properties of whole-cell Ca(2+) channel currents, and of non-L-type Ca(2+) channel currents between the neurons from these two culture systems. Our data suggested that the difference of whole-cell Ca(2+) currents may result from the differences in non-L-type currents. Understanding of these properties will considerably advance studies of VGCCs in neurons from pure or mixed culture.

Fig. 1

The morphological and membrane capacitance of neurons in pure and mixed culture. Typical bipolar neurons (indicated by arrow) from pure (left) and mixed (right) culture were selected (arrows; a). The membrane capacitances in pure (n = 17) and mixed (n = 35) neurons were not different (b)

Fig. 2

The whole-cell Ca²⁺ currents in pure and mixed cultures. The neurons were hold at −80 mV and then depolarized to elicit the whole cell Ca²⁺ channel currents (a) in the pure (upon) and mixed (below) cultured neurons. There were significant differences in current densities at voltage ranges from −30 to +10 mV in pure (circle; n = 17) and mixed (triangle; n = 35) cultured neurons (b). *P < 0.05 and **P < 0.01 as compared with pure cultured neurons (students t test)

Fig. 3

Activation properties of VGCCs in pure and mixed cultured neurons. The voltage-dependent activation curves were fitted by Boltzman equation (a) and showed significantly different half-activation potential (b) and slope factor (c) between pure (circle; n = 17) and mixed (triangle; n = 35) cultured neurons. **P < 0.01 as compared with pure cultured neurons (students t test)

Fig. 4

LCC current and non-L-currents in pure and mixed cultured neurons. The LCC currents were dissociated by nifedipine (10 μM) from whole cell Ca²⁺ currents (a). The percentage of LCC currents in whole cell currents from mixed cultured neurons (triangle; n = 12) was higher than from pure (circle; n = 6) cultured neurons (b). The current densities were not different for L-type current (c), but were significantly different for non-L-type currents (d) between pure and mixed cultured neurons. *P < 0.05 and **P < 0.01 as compared with pure cultured neurons (students t test)

Fig. 5

Activation properties of L-type currents and non-L-type currents in pure and mixed cultured neurons. The voltage-dependent activation curves of L-type current (a) and non-L-currents (b) were fitted by Boltzman equation and showed that both of half-activation potential (c) and slope factor (d) were significantly different between pure (circle; n = 6) and mixed (triangle; n = 10) cultured neurons in the non-L-type currents. *P < 0.05 and **P < 0.01 as compared with pure cultured neurons (students t test)