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INNOVATIVE METHODOLOGY

Presynaptic Calcium Measurements at Physiological Temperatures Using a New Class of Dextran-Conjugated Indicators

¹Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115; and ²Invitrogen, Corporation, Eugene, Oregon 97402

Submitted 16 January 2004; accepted in final form 26 February 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.

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