Abstract
In developing neurons the frequency of long duration, spontaneous, transient calcium (Ca2+) 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 Ca2+ transients with slow kinetics, called Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ waves. In some experiments, we determined the temporal correlation between Ca2+ 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 Ca2+ waves and axon growth dynamics is disrupted by ethanol.
Highlights
Maternal consumption of alcohol during pregnancy can produce abnormalities in the central nervous system of the fetus, resulting in life-long cognitive and behavioral impairments called fetal alcohol spectrum disorder (FASD) [1]
We found that chronic exposure to or 87 mM ethanol in the medium of low-density cultures of embryonic rat hippocampal pyramidal neurons delays initial axon outgrowth and disrupts axon growth dynamics [11]
To determine whether Ca2+ waves occur in dissociated hippocampal neurons, we loaded fetal rat hippocampal neurons with the Ca2+ indicator dye Fluo-3 and imaged their axonal growth cones for min
Summary
Maternal consumption of alcohol during pregnancy can produce abnormalities in the central nervous system of the fetus, resulting in life-long cognitive and behavioral impairments called fetal alcohol spectrum disorder (FASD) [1]. Spontaneous Ca2+ spikes in axonal growth cones of Syrian hamster cortical neurons in vitro regulate axon branching as well as axon growth [17]. In both types of neurons, the frequency of these Ca2+ transients is inversely correlated with the rate of axon elongation, and increased frequency is characteristic of stationary growth cones. We used time-series Fluo-3 imaging of growth cone Ca2+ to assess effects of ethanol on slow, spontaneous Ca2+ waves in hippocampal pyramidal neurons under the same conditions that alter axon growth dynamics. We show for the first time that ethanol alters the incidence of these Ca2+ waves in axonal growth cones and disrupts their relationship to axon extension
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