Timing of neuronal and glial ultrastructure disruption during brain slice preparation and recovery in vitro
Timing of neuronal and glial ultrastructure disruption during brain slice preparation and recovery in vitro
复制标题
DOI：
10.1002/cne.10825
复制
发表时间：
2003-10-06
影响因子：
2.5
通讯作者：
Harris, KM
中科院分区：
文献类型：
--
作者：
Fiala, JC;Kirov, SA;Harris, KM
海马切片通常比灌注固定的海马有更多的突触，但这种突触发生的原因尚不清楚。在21日龄大鼠切片制备过程中突触触发器的超微结构证据进行了研究。切片切碎在温暖或寒冷的条件下，并固定后0，5，25，60，或180分钟的孵育在一个接口室进行了比较，与固定的海马灌注或浸泡的整个海马。与灌注固定的海马相比，这些切片中没有显著的突触发生，但在体外切片制备和恢复过程中有其他结构变化。整个海马和切片在温暖的条件下制备的轴突包被囊泡的增加，表明广泛的神经递质释放。糖原颗粒在0分钟切片中从星形胶质细胞和神经元中耗尽，1小时开始重新出现，3小时完全恢复。树突状微管最初在切片中分解，但在5分钟后重新组装成正常的轴向阵列。微管在5分钟时较短（12.3 +/- 1.1 μ m），但与灌注固定的海马（91 +/- 22 μ m）相比，微管在3小时时恢复正常长度（84.6 +/- 20.0 μ m）。孵育后5分钟和25分钟，分别在15 +/- 3%和9 +/- 4%的树突棘中短暂出现微管。灌注固定的海马和3小时切片中不存在棘微管。在阻断活性的培养基中进行冰冷解剖和振动解剖，最初产生较少的糖原损失、包被囊泡和微管解体。在34 ℃下将这些切片浸没在正常的含氧培养基中导致糖原耗尽，以及在1分钟内增加的包被囊泡和微管分解。(C)2003 Wiley-Liss，Inc.
Hippocampal slices often have more synapses than perfusion-fixed hippocampus, but the cause of this synaptogenesis is unclear. Ultrastructural evidence for synaptogenic triggers during slice preparation was investigated in 21-day-old rats. Slices chopped under warm or chilled conditions and fixed after 0, 5, 25, 60, or 180 minutes of incubation in an interface chamber were compared with hippocampi fixed by perfusion or by immersion of the whole hippocampus. There was no significant synaptogenesis in these slices compared with perfusion-fixed hippocampus, but there were other structural changes during slice preparation and recovery in vitro. Whole hippocampus and slices prepared under warm conditions exhibited an increase in axonal coated vesicles, suggesting widespread neurotransmitter release. Glycogen granules were depleted from astrocytes and neurons in 0-min slices, began to reappear by 1 hour, and had fully recovered by 3 hours. Dendritic microtubules were initially disassembled in slices, but reassembled into normal axial arrays after 5 minutes. Microtubules were short at 5 minutes (12.3 +/- 1.1 mum) but had recovered normal lengths by 3 hours (84.6 +/- 20.0 mum) compared with perfusion-fixed hippocampus (91 +/- 22 mum). Microtubules appeared transiently in 15 +/- 3% and 9 +/- 4% of dendritic spines 5 and 25 minutes after incubation, respectively. Spine microtubules were absent from perfusion-fixed hippocampus and 3-hour slices. Ice-cold dissection and vibratomy in media that blocked activity initially produced less glycogen loss, coated vesicles, and microtubule disassembly. Submersing these slices in normal oxygenated media at 34degreesC led to glycogen depletion, as well as increased coated vesicles and microtubule disassembly within 1 minute. (C) 2003 Wiley-Liss, Inc.