Presynaptic calcium measurements at physiological temperatures using a new class of dextran-conjugated indicators

被引:31
作者
Beierlein, M
Gee, KR
Martin, VV
Regehr, WG
机构
[1] Harvard Univ, Sch Med, Dept Neurobiol, Boston, MA 02115 USA
[2] Invitrogen Corp, Eugene, OR 97402 USA
关键词
D O I
10.1152/jn.00057.2004
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
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.
引用
收藏
页码:591 / 599
页数:9
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