Beierlein Michael, Gee Kyle R, Martin Vladimir V, Regehr Wade G
Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115, USA.
J Neurophysiol. 2004 Jul;92(1):591-9. doi: 10.1152/jn.00057.2004.
Presynaptic calcium (Ca(pre)) has been studied extensively because of its role in triggering and modulating neurotransmitter release. Although calcium regulation and calcium-driven processes can be strongly temperature dependent, technical difficulties have limited most studies of Ca(pre) to temperatures well below the physiological range. Here we assessed the use of membrane-permeant acetoxymethyl (AM) indicators and dextran-conjugated indicators for measuring Ca(pre) at physiological temperatures. A comparison of these two types of indicators loaded into parallel fibers of rat cerebellar slices revealed striking differences. AM indicators were rapidly extruded from axons and presynaptic terminals and therefore cannot be used for long-term measurements at high temperatures. In contrast, dextran-conjugated indicators were retained within parallel fibers and are therefore well suited to measuring Ca(pre) at physiological temperatures. The limited number of dextran indicators available prompted us to synthesize three new indicators that show peak emission in the red (575-600 nm). These indicators allow for simultaneous use of multiple calcium indicators that can be readily distinguished on the basis of excitation and emission wavelengths, use of excitation and emission wavelengths that are relatively insensitive to tissue autofluorescence, and measurements in systems with expression of green fluorescent protein (GFP). Thus we find that dextran-conjugated indicators are well suited to long-term recordings of Ca(pre) at physiological temperatures and that the development of new red indicators greatly extends their utility.
突触前钙（Ca(pre)）因其在触发和调节神经递质释放中的作用而受到广泛研究。尽管钙调节和钙驱动的过程可能强烈依赖于温度，但技术难题限制了大多数关于Ca(pre)的研究只能在远低于生理范围的温度下进行。在此，我们评估了使用膜通透性乙酰氧基甲基（AM）指示剂和葡聚糖偶联指示剂在生理温度下测量Ca(pre)的情况。对加载到大鼠小脑切片平行纤维中的这两种指示剂进行比较，发现了显著差异。AM指示剂会迅速从轴突和突触前终末排出，因此不能用于高温下的长期测量。相比之下，葡聚糖偶联指示剂能保留在平行纤维内，因此非常适合在生理温度下测量Ca(pre)。可用葡聚糖指示剂数量有限促使我们合成了三种新的指示剂，它们在红色区域（575 - 600 nm）呈现发射峰。这些指示剂允许同时使用多种钙指示剂，基于激发和发射波长可轻易区分它们，使用对组织自发荧光相对不敏感的激发和发射波长，并能在表达绿色荧光蛋白（GFP）的系统中进行测量。因此我们发现葡聚糖偶联指示剂非常适合在生理温度下对Ca(pre)进行长期记录，并且新红色指示剂的开发极大地扩展了它们的用途。
