Synaptic Vesicles: Test for a Role in Presynaptic Calcium Regulation

Abstract

Membrane-bound organelles such as mitochondria and the endoplasmic reticulum play an important role in neuronal

In VivoPerformance of Genetically Encoded Indicators of Neural Activity in Flies

“…2, available at www.jneurosci.org as supplemental material). The missing steady state is in contrast to the reported time course of presynaptic calcium in this preparation measured with synthetic dyes (Macleod et al, 2002(Macleod et al, , 2004 during prolonged stimulation periods. At 40 Hz stimulation, no such transient peak is visible.…”

“…A narrow dynamic range would be expected for both indicators from their large Hill coefficient of n ϭ 3.3 [GCaMP1.3 (Nakai et al, 2001)] and n ϭ 3.8 [GCaMP1.6 (Nakai, personal communication)]. Moreover, we expected saturation of the GCaMPs in the experiments in Figure 3 because calcium changes of many hundred nanomoles up to the micromolar range can be expected in the Drosophila boutons under these conditions (Macleod at al., 2004), and K d values of 235 and 146 nM have been reported for GCaMP1.3 (Nakai et al, 2001) and GCaMP1.6, respectively, when measured in vitro (Nakai, personal communication). This discrepancy cannot be explained by the interplay of several compensating factors (such as cooperative calcium binding, saturation of the probe, missing background subtraction, and bleaching) because a linear relationship of the GCaMP1.3 fluorescence to neural activity was observed (Fig.…”

“…When fast synthetic dyes (Macleod et al, 2002(Macleod et al, , 2004 were used, steady-state fluorescence signals with linear relationship to the stimulus frequency were observed in the same preparation. The described signals in Figure 3 and the linear relationship of GCaMP1.3 and YC3.3 signals to neural activity are in accordance with these results.…”

“…Conversely, the SNR increased with the expression level, pointing out an important tradeoff between signal kinetics and SNR. Using rather low concentrations of Oregon-Green-BAPTA with a K d of ϳ500 nM, Macleod et al (2002Macleod et al ( , 2004 showed that the decay in calcium after a single AP as well as after long stimulus trains was ϳ60 ms in type Ib boutons of muscle 6/7 Drosophila NMJs. Thus, 60 ms is likely to correspond to the calcium extrusion rate, a value that we could not approach because the fastest time constant of the decay was 350 ms reported by GCaMP1.6.…”

“…All experiments were performed in HL6 (Macleod et al, 2002) containing 7 mM glutamate and 1.5 mM calcium, unless otherwise stated. Glutamate effectively blocked postsynaptic muscle contractions at concentrations Ն5 mM without influencing presynaptic Ca dynamics (Macleod et al, 2004). In brief, late third-instar larvae were pinned to the bottom of a self-made recording chamber and opened up along the dorsal midline, and the trachea, gut, and fat body were removed.…”