Calcium (Ca2+) channels are sensitive to ethanol and Ca2+ signaling is a critical regulator of axonal growth and guidance. Effects of acute and chronic exposure to ethanol (22, 43, or 87 mM) on voltage-gated Ca2+ channels (VGCCs) in whole cells, and KCl-induced Ca2+ transients in axonal growth cones, were examined using dissociated hippocampal cultures. Whole-cell patch-clamp analysis in neurons with newly-formed axons (Stage 3) revealed that rapidly inactivating, low-voltage activated (LVA) and non-inactivating, high-voltage activated (HVA) currents were both inhibited in a dose-dependent manner by acute ethanol, with relatively greater inhibition of HVA currents. When assessed by Fluo-4-AM imaging, baseline fluorescence and Ca2+ response to ethanol in Stage 3 neurons was similar compared to neurons without axons, but peak Ca2+ transient amplitudes in response to bath-applied KCl were greater in Stage 3 neurons and were decreased by acute ethanol. The amplitude of Ca2+ transients elicited specifically in axonal growth cones by focal application of KCl was also inhibited by acute exposure to moderate-to-high concentrations of ethanol (43 or 87 mM), whereas a lower concentration (22 mM) had no effect. When 43 or 87 mM ethanol was present continuously in the medium, KCl-evoked Ca2+ transient amplitudes were also reduced in growth cones. In contrast, Ca2+ transients were increased by continuous exposure to 22 mM ethanol. Visualization using a fluorescent dihydropyridine analog revealed that neurons continuously exposed to ethanol expressed increased amounts of L-type Ca2+ channels, with greater increases in axonal growth cones than cell bodies. Thus, acute ethanol reduces Ca2+ current and KCl-induced Ca2+ responses in whole cells and axonal growth cones, respectively, and chronic exposure is also generally inhibitory despite apparent up-regulation of L-type channel expression. These results are consistent with a role for altered growth cone Ca2+ signaling in abnormal neuromorphogenesis associated with fetal alcohol spectrum disorders.
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