Fiala John C, Kirov Sergei A, Feinberg Marcia D, Petrak Lara J, George Priya, Goddard C Alex, Harris Kristen M
Department of Biology, Boston University, Boston, Massachusetts 02215, USA.
J Comp Neurol. 2003 Oct 6;465(1):90-103. doi: 10.1002/cne.10825.
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 microm) but had recovered normal lengths by 3 hours (84.6 +/- 20.0 microm) compared with perfusion-fixed hippocampus (91 +/- 22 microm). 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 34 degrees C led to glycogen depletion, as well as increased coated vesicles and microtubule disassembly within 1 minute.
海马切片通常比灌注固定的海马具有更多的突触，但这种突触形成的原因尚不清楚。在21日龄大鼠中研究了切片制备过程中突触形成触发因素的超微结构证据。将在温暖或寒冷条件下切碎并在界面室中孵育0、5、25、60或180分钟后固定的切片与通过灌注或整个海马浸泡固定的海马进行比较。与灌注固定的海马相比，这些切片中没有明显的突触形成，但在切片制备和体外恢复过程中存在其他结构变化。在温暖条件下制备的整个海马和切片显示轴突包被小泡增加，提示广泛的神经递质释放。糖原颗粒在0分钟切片的星形胶质细胞和神经元中耗尽，1小时后开始重新出现，3小时后完全恢复。树突微管最初在切片中解体，但5分钟后重新组装成正常的轴向排列。与灌注固定的海马（91±22微米）相比，微管在5分钟时较短（12.3±1.1微米），但3小时后恢复到正常长度（84.6±20.0微米）。孵育5分钟和25分钟后，分别有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小时切片中没有棘微管。在阻断活性的培养基中进行冰冷解剖和振动切片最初产生的糖原损失、包被小泡和微管解体较少。将这些切片浸入34℃的正常含氧培养基中1分钟内导致糖原耗尽，以及包被小泡增加和微管解体。
