Klose M K, Boulianne G L, Robertson R M, Atwood H L
Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
J Neurophysiol. 2009 Aug;102(2):901-13. doi: 10.1152/jn.91209.2008. Epub 2009 May 27.
Maintenance of synaptic transmission requires regulation of intracellular Ca(2+) in presynaptic nerve terminals; loss of this regulation at elevated temperatures may cause synaptic failure. Accordingly, we examined the thermosensitivity of presynaptic calcium regulation in Drosophila larval neuromuscular junctions, testing for effects of disrupting calcium clearance. Motor neurons were loaded with the ratiometric Ca(2+) indicator Fura-dextran to monitor calcium regulation as temperature increased. Block of the Na(+)/Ca(2+) exchanger or removal of extracellular Ca(2+) prevented the normal temperature-induced increase in resting calcium. Conversely, two treatments that interfered with Ca(2+) clearance-inactivation of the endoplasmic reticulum Ca(2+)-ATPase with thapsigargin and inhibition of the plasma membrane Ca(2+)-ATPase with high pH-significantly accelerated the temperature-induced rise in resting Ca(2+) concentration and reduced the thermotolerance of synaptic transmission. Disrupting Ca(2+)-ATPase function by interfering with energy production also facilitated the temperature-induced rise in resting [Ca(2+)] and reduced thermotolerance of synaptic transmission. Conversely, fortifying energy levels with extra intracellular ATP extended the operating temperature range of both synaptic transmission and Ca(2+) regulation. In each of these cases, Ca(2+) elevations evoked by an electrical stimulation of the nerve (evoked Ca(2+) responses) failed when resting Ca(2+) remained >e 200 nM for several minutes. Failure of synaptic function was correlated with the release of intracellular calcium stores, and we provide evidence suggesting that release from the mitochondria disrupts evoked calcium responses and synaptic transmission. Thus the thermal limit of synaptic transmission may be directly linked to the stability of ATP-dependent mechanisms that regulate intracellular ion concentrations in the nerve terminal.
突触传递的维持需要对突触前神经末梢内的细胞内钙离子（Ca(2+)）进行调节；在高温下这种调节的丧失可能会导致突触功能衰竭。因此，我们研究了果蝇幼虫神经肌肉接头处突触前钙调节的热敏感性，测试了破坏钙清除的影响。运动神经元被装载了比率型Ca(2+)指示剂Fura-葡聚糖，以监测随着温度升高的钙调节情况。钠/钙交换体的阻断或细胞外钙的去除可防止正常温度诱导的静息钙增加。相反，两种干扰Ca(2+)清除的处理——用毒胡萝卜素使内质网Ca(2+)-ATP酶失活以及用高pH抑制质膜Ca(2+)-ATP酶——显著加速了温度诱导的静息Ca(2+)浓度升高，并降低了突触传递的耐热性。通过干扰能量产生来破坏Ca(2+)-ATP酶功能也促进了温度诱导的静息[Ca(2+)]升高，并降低了突触传递的耐热性。相反，用额外的细胞内ATP增强能量水平扩展了突触传递和Ca(2+)调节的工作温度范围。在这些情况中的每一种，当静息Ca(2+)在几分钟内保持>200 nM时，神经电刺激诱发的Ca(2+)反应（诱发Ca(2+)反应）均失败。突触功能的失败与细胞内钙储存的释放相关，并且我们提供的证据表明线粒体释放会破坏诱发的钙反应和突触传递。因此，突触传递的热极限可能直接与调节神经末梢内细胞内离子浓度的ATP依赖机制的稳定性相关。
