Ethanol withdrawal influences survival and morphology of developing rat hippocampal neurons in vitro

Previous studies in this laboratory have shown that, like their counterparts in vivo, fetal rat hippocampal pyramidal neurons in culture develop abnormally small dendritic arbors when exposed to ethanol. This study asked whether ethanol's inhibitory effects on dendritic development differ when the duration of ethanol exposure and timing of withdrawal are varied to correspond with early versus later stages of development and whether ethanol withdrawal influences survival of these neurons.

We compared neurons exposed continuously for 6 or 14 days to ethanol (70 mM) with neurons transferred from ethanol-containing medium to control medium either 1 day after adding ethanol (before dendrites elongated) or 6 days after adding ethanol (after dendrites began elongating). We then performed morphometric and cell density analyses at 6 and 14 days using digital images of neurons immunostained with microtubule-associated protein 2 (MAP2) to visualize dendrites.

Continuous exposure to ethanol decreased the length and number of dendrites formed but had no effect on neuron survival compared with controls without ethanol. Dendritic length was less inhibited when ethanol was withdrawn after 1 day, but the number of dendrites per cell was unchanged compared with neurons continuously exposed to ethanol. Withdrawal from ethanol at 1 day slightly enhanced the survival of neurons assessed at 14 days compared with neurons in control medium and with neurons exposed continuously to ethanol. In contrast, withdrawal from ethanol at 6 days severely decreased the number of neurons at 14 days.

These results suggest that dendrites can achieve normal length when ethanol exposure is limited to only 1 day and withdrawal occurs before dendrites begin elongating. However, a persistent reduction in dendrite number results in smaller overall dendritic arbor size. Although continuous exposure to ethanol has little effect on neuron survival in these cultures, and exposure limited to 1 day followed by withdrawal can be neuroprotective against cell death associated with increased time in culture, longer exposure before withdrawal can trigger cell death.