Yamane K, Yokono K, Okada Y
Department of Physiology, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ko, 650-0017, Kobe, Japan.
J Neurosci Methods. 2000 Nov 30;103(2):163-71. doi: 10.1016/s0165-0270(00)00312-5.
To investigate the functional significance of anaerobic and aerobic glycolysis on neural activity and levels of high energy phosphates, we tested the effects of glucose, mannose, fructose lactate, and pyruvate on the maintenance of neural activity and on the levels of ATP and creatine-P (CrP) in hippocampal slices. For an index of neural activity, population spikes (PS) evoked in the granule cell layer were monitored. Immediately after deprivation of glucose, the PS amplitude was gradually reduced and extinguished within 30 mm. Replacement of glucose with either lactate or pyruvate resulted in a decay and loss of PS with a similar time-course as observed during glucose deprivation. However, after the complete loss of neural activity for 10-20 min the PS reappeared and recovered to normal levels. The replacement of glucose with either mannose or fructose resulted in a transient decrease of the PS to 80-70% of the original amplitude in 20 mm, followed by recovery. The time-course of the decrease of PS in the mannose-containing medium was slower than that in the medium containing fructose and the time-course of recovery was faster. ATP and CrP were reduced to 90 and 70% of original level in each slice after glucose deprivation for 30 and 100 mm, respectively. In media containing either lactate, pyruvate, mannose, or fructose, the level of ATP and CrP was maintained at the original level. The anaerobic metabolic rate of glucose, mannose and fructose, determined by the rate of lactate production during complete anoxia, was consistent with the order of the decay and recovery of the PS in mannose and fructose-containing medium. The mode of the transient decay or loss of PS with no apparent reduction in the levels of ATP and CrP in each slice, in the presence of either mannose, fructose or lactate, indicates that anaerobic glycolysis is crucial for the maintenance of PS. The results obtained in this experiment are not in accordance with the reports by Schurr and Fowler in which they showed that lactate can support neural activity, although they did not measure the levels of ATP and CrP in slices. The present experiment disclosed that this discrepancy was due to the difference of slice preparation; namely rapidly prepared slice with shorter period anoxia (1 mm after removal of hippocampal tissue block) gives the results mentioned in the present study whereas slowly prepared slices by vibratome or chopper method with longer period of ischemia (5-20 mm), did not show transient loss of PS after application of lactate. Thus present experiment indicates that glycolytic process is essential for maintaining neural activity for physiological state of slices, if it is admitted to say that rapidly prepared slices is more physiological because they are exposed on shorter period of ischemia, and that more careful attention should be paid for the interpretation of the results of slice experiment according to the method of slice preparation.
为了研究无氧糖酵解和有氧糖酵解对神经活动及高能磷酸盐水平的功能意义，我们测试了葡萄糖、甘露糖、果糖、乳酸和丙酮酸对海马切片中神经活动维持以及三磷酸腺苷（ATP）和磷酸肌酸（CrP）水平的影响。作为神经活动的指标，我们监测了颗粒细胞层诱发的群体峰电位（PS）。葡萄糖剥夺后，PS波幅立即逐渐降低，并在30分钟内消失。用乳酸或丙酮酸替代葡萄糖后，PS出现衰减和消失，其时间进程与葡萄糖剥夺时观察到的相似。然而，在神经活动完全丧失10 - 20分钟后，PS重新出现并恢复到正常水平。用甘露糖或果糖替代葡萄糖会导致PS在20分钟内短暂降低至原始波幅的80 - 70%，随后恢复。含甘露糖培养基中PS降低的时间进程比含果糖培养基中的慢，而恢复的时间进程更快。葡萄糖剥夺30分钟和100分钟后，每片切片中的ATP和CrP分别降至原始水平的90%和70%。在含有乳酸、丙酮酸、甘露糖或果糖的培养基中，ATP和CrP水平维持在原始水平。通过完全缺氧时乳酸生成速率测定的葡萄糖、甘露糖和果糖的无氧代谢率，与含甘露糖和果糖培养基中PS衰减和恢复的顺序一致。在存在甘露糖、果糖或乳酸的情况下，PS短暂衰减或消失但每片切片中ATP和CrP水平无明显降低，这表明无氧糖酵解对于PS的维持至关重要。本实验所得结果与舒尔和福勒的报告不一致，他们在报告中表明乳酸可以支持神经活动，尽管他们没有测量切片中的ATP和CrP水平。本实验揭示这种差异是由于切片制备方法的不同；即快速制备的切片缺氧时间较短（海马组织块取出后1分钟）得到了本研究中提到的结果，而通过振动切片机或切片刀方法缓慢制备的切片缺血时间较长（5 - 20分钟），在应用乳酸后未显示PS的短暂丧失。因此，本实验表明，如果可以说快速制备的切片更具生理性，因为它们暴露于较短的缺血时间，那么糖酵解过程对于维持切片生理状态下的神经活动至关重要，并且对于根据切片制备方法解释切片实验结果应给予更多关注。
