Ethanol Modulates Spontaneous Calcium Waves in Axonal Growth Cones in Vitro

In developing neurons the frequency of long duration, spontaneous, transient calcium (Ca(2+)) elevations localized to the growth cone, is inversely related to the rate of axon elongation and increases several fold when axons pause. Here we report that these spontaneous Ca(2+) transients with slow kinetics, called Ca(2+) waves, are modulated by conditions of ethanol exposure that alter axonal growth dynamics. Using time-series fluorescence calcium imaging we found that acute treatment of fetal rat hippocampal neurons with 43 or 87 mM ethanol at an early stage of development in culture decreased the percent of axon growth cones showing at least one Ca(2+) wave during 10 min of recording, from 18% in controls to 5% in cultures exposed to ethanol. Chronic exposure to 43 mM ethanol also reduced the incidence of Ca(2+) waves to 8%, but exposure to 87 mM ethanol increased their incidence to 31%. Neither chronic nor acute ethanol affected the peak amplitude, time to peak or total duration of Ca(2+) waves. In some experiments, we determined the temporal correlation between Ca(2+) waves and growth and non-growth phases of axonal growth dynamics. As expected, waves were most prevalent in stationary or retracting growth cones in all treatment groups, except in cultures exposed chronically to 87 mM ethanol. Thus, the relationship between growth cone Ca(2+) waves and axon growth dynamics is disrupted by ethanol.