Lindsley Tara A, Clarke Shannon
Center for Neuropharmacology and Neuroscience, Albany Mediccal College, Albany, New York, USA.
Alcohol Clin Exp Res. 2004 Jan;28(1):85-92. doi: 10.1097/01.ALC.0000106306.60134.C1.
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 Dendritic length was less inhibited when ethanol was withdrawn after 1 day, but the number of dendrites per cell was unchanged compared with neurons continuously exposed to ethanol.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.
本实验室之前的研究表明，与体内情况类似，培养的胎鼠海马锥体神经元在接触乙醇时会发育出异常小的树突分支。本研究旨在探讨当乙醇暴露持续时间和撤药时间与发育的早期和晚期阶段相对应时，乙醇对树突发育的抑制作用是否存在差异，以及乙醇撤药是否会影响这些神经元的存活。
我们将连续6天或14天暴露于乙醇（70 mM）的神经元与在添加乙醇后1天（树突伸长前）或6天（树突开始伸长后）从含乙醇培养基转移至对照培养基的神经元进行比较。然后，在第6天和第14天，使用用微管相关蛋白2（MAP2）免疫染色的神经元数字图像进行形态计量学和细胞密度分析，以观察树突。
与未接触乙醇的对照组相比，持续暴露于乙醇会减少形成的树突长度和数量，但对神经元存活没有影响。在1天后撤去乙醇时，树突长度受到的抑制较小，但与持续暴露于乙醇的神经元相比，每个细胞的树突数量没有变化。与对照培养基中的神经元以及持续暴露于乙醇的神经元相比，在1天撤去乙醇会略微提高在第14天评估的神经元存活率。相比之下，在6天撤去乙醇会严重减少第14天的神经元数量。
这些结果表明，当乙醇暴露仅限于1天且在树突开始伸长之前撤药时，树突可以达到正常长度。然而，树突数量的持续减少会导致整体树突分支大小变小。尽管持续暴露于乙醇对这些培养物中的神经元存活影响不大，并且暴露1天然后撤药可以对与培养时间延长相关的细胞死亡起到神经保护作用，但撤药前较长时间的暴露会引发细胞死亡。
