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Calcium Dynamics, Buffering, and Buffer Saturation in the Boutons




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Calcium Dynamics, Buffering, and Buffer Saturation in the Boutons of Dentate Granule-Cell Axons in the Hilus Meyer B. Jackson and Stephen J. Redman Division of Neuroscience, John Curtin School of Medical Research, Canberra, ACT 0200, Australia The axons of dentate gyrus granule cells form synapses in the hilus. Ca 2 signaling was investigated in the boutons of these axons using confocal fluorescence imaging. Boutons were loaded with various concentrations of the Ca 2 indicator Oregon Green BAPTA-1 by patch-clamping the cell bodies and allowing the dye to diffuse into the axon. Resting free [Ca 2] started at 74 nM, rose to 1 M immediately after an action potential, and then decayed to rest with a time constant of 43 msec (all extrapolated to a dye concentration of zero). Action potential-induced [Ca 2] rises were smaller in larger boutons, consistent with a size-independent Ca 2 channel density of 45/m 2. Action potential-induced [Ca 2] changes varied with dye concentration in a manner consistent with E20 for the ratio of endogenous buffer-bound Ca 2 to free Ca 2. During trains of action potentials, [Ca 2] increments summed supralinearly by more than that expected from dye saturation. The amount of endogenous Ca 2 buffering declined as [Ca 2] rose, and this saturation indicated a buffer with a dissociation constant of500 nM and a concentration of130M. This is similar to the dissociation constant of calbindin- D28K, a Ca 2-binding protein that is abundant in dentate granule cells. Thus, calbindin-D28K is a good candidate for the Ca 2 buffer revealed by these experiments. The saturation of endogenous buffer can generate short-term facilitation by amplifying [Ca 2] changes during repetitive activity. Buffer saturation may also be relevant to the presynaptic induction of long-term potentiation at synapses formed by dentate granule cells. Key words: nerve terminals; calcium dynamics; calcium buffers; hippocampus; dentate gyrus; calbindin-D28K; mossy fibers Introduction Calcium ions enter a nerve terminal during presynaptic action potentials and bind to Ca 2 sensors to trigger neurotransmitter release. Cytoplasmic Ca 2 buffers compete for this Ca 2 to dampen the Ca 2 rise; Ca 2 sequestration and extrusion ma- chinery restore Ca 2 to resting levels. By shaping the Ca 2 signal, the Ca 2-regulating systems of a nerve terminal play an impor- tant role in controlling synaptic function. Cytoplasmic Ca 2 sig- naling can be investigated by imaging with fluorescent dyes. Most previous fluorometric studies of presynaptic Ca 2 fall into two distinct groups. Large nerve terminals are loaded by direct injec- tion of Ca 2-sensitive dye (Smith et al., 1988; Jackson et al., 1991; Helmchen et al., 1997), affording a degree of control over the cytoplasm and membrane. This method cannot be applied to the far more abundant nerve terminals of smaller size. Alternatively, small nerve terminals can be loaded by extracellular application of membrane-permeable dyes (Regehr and Tank, 1991a; Mel- amed et al., 1993; Umbach et al., 1998). This overcomes the size obstacle, but the dye concentration is difficult to control. A promising new approach ...





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