Kirov S A, Petrak L J, Fiala J C, Harris K M
Department of Neurosurgery, Human Brain Laboratory, Medical College of Georgia, 1120 15th Street, CB-2607, Augusta, GA 30912, USA.
Neuroscience. 2004;127(1):69-80. doi: 10.1016/j.neuroscience.2004.04.053.
More dendritic spine synapses occur on mature neurons in hippocampal slices by 2 h of incubation in vitro, than in perfusion-fixed hippocampus. What conditions initiate this spinogenesis and how rapidly do the spines begin to proliferate on mature neurons? To address these questions, CA1 field of the hippocampus neurons expressing green fluorescent protein in living slices from mature mice were imaged with two-photon microscopy. Spines disappeared and dendrites were varicose immediately after slice preparation in ice-cold artificial cerebrospinal fluid (ACSF). Electron microscopy (EM) revealed disrupted dendritic cytoplasm, enlarged or free-floating postsynaptic densities, and excessive axonal endocytosis. Upon warming dendritic varicosities shrank and spines rapidly reappeared within a few minutes illustrating the remarkable resilience of mature hippocampal neurons in slices. When membrane impermeant sucrose was substituted for NaCl in ACSF dendrites remained spiny at ice-cold temperatures and EM revealed less disruption. Nevertheless, spine number and length increased within 30 min in warm ACSF even when the extracellular calcium concentration was zero and synaptic transmission was blocked. When slices were first recovered for several hours and then chilled in 6 degrees C ACSF many spines disappeared and the dendrites became varicose. Upon re-warming varicosities shrank and spines reemerged in the same position from which they disappeared. In addition, new spines formed and spines were longer suggesting that chilling, not the initial injury from slicing, caused the spines to disappear while re-warming triggered the spine proliferation on mature neurons. The new spines might be a substrate for neuronal recovery of function, when neurons have been chilled or exposed to other traumatic conditions that disrupt ionic homeostasis.
在体外孵育2小时后，海马切片中成熟神经元上出现的树突棘突触比灌注固定的海马中的更多。是什么条件引发了这种棘突形成，并且棘突在成熟神经元上开始增殖的速度有多快？为了解决这些问题，使用双光子显微镜对来自成年小鼠的活体切片中表达绿色荧光蛋白的海马神经元的CA1区进行成像。在冰冷的人工脑脊液（ACSF）中切片制备后，棘突立即消失，树突出现静脉曲张。电子显微镜（EM）显示树突细胞质破坏、突触后致密物增大或游离、轴突内吞作用过度。当变暖时，树突静脉曲张收缩，棘突在几分钟内迅速重新出现，这说明了切片中成熟海马神经元具有显著的恢复能力。当在ACSF中用膜不透性蔗糖替代NaCl时，树突在冰冷温度下仍有棘突，并且EM显示破坏较少。然而，即使细胞外钙浓度为零且突触传递被阻断，在温暖的ACSF中30分钟内棘突数量和长度仍会增加。当切片先恢复数小时，然后在6摄氏度的ACSF中冷却时，许多棘突消失，树突变得静脉曲张。再次变暖时，静脉曲张收缩，棘突从它们消失的相同位置重新出现。此外，新的棘突形成且棘突更长，这表明冷却而非切片的初始损伤导致棘突消失，而再次变暖触发了成熟神经元上的棘突增殖。当神经元受到冷却或暴露于其他破坏离子稳态的创伤条件时，新的棘突可能是神经元功能恢复的基础。
