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. 2019 Mar 27;39(13):2416–2429. doi: 10.1523/JNEUROSCI.3068-18.2019

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Copyright © 2019 Gratz et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 2. — Endogenous tagging of Cacophony does not perturb presynaptic Ca²⁺ influx or synaptic function. A, Live fluorescence images of type-Ib boutons from a cac^sfGFP-N motor terminal showing Cac^sfGFP-N (top) and AF647-dextran (center), which was co-loaded with rhod-dextran (image not shown). B, Ratiometric fluorescence changes (rhod-dextran relative to AF647-dextran) in presynaptic motorneuron terminals during stimulation in wild type and cac^sfGFP-N. An action potential was initiated every second, for a period of 10 s, followed by a 1 s, 20 Hz train of action potentials. C, Plot of average Ca²⁺ levels (R) in terminals before nerve stimulation for type-Ib and -Is terminals in wild-type and cac^sfGFP-N boutons [wild-type Ib, 2.74 ± 0.44, one NMJ from each of 5 animals (N = 5); cac^sfGFP-N Ib, 2.47 ± 0.49, N = 8, p = 0.69; wild-type Is, 2.16 ± 0.47, N = 5; cac^sfGFP-N Is, 2.94 ± 0.32, N = 6, p = 0.21; Student's t test). D, Plot of the average amplitude of single action potential-mediated Ca²⁺ transients [ΔR; wild-type Ib, 4.50 ± 0.81, one NMJ from each of 5 animals (N = 5); cac^sfGFP-N Ib, 3.90 ± 1.07, N = 8, p = 0.67; wild-type Is, 6.28 ± 1.79, N = 5; cac^sfGFP-N Is, 7.33 ± 2.20, N = 6, p = 0.72; Student's t test]. E, Plot of the average amplitude of 1 s, 20 Hz action potential train-mediated Ca²⁺ transients [wild-type Ib, 9.71 ± 1.53, one NMJ from each of 5 animals (N = 5); cac^sfGFP-N Ib, 8.13 ± 2.21, N = 8, p = 0.57; wild-type Is, 12.26 ± 3.36, N = 5; cac^sfGFP-N Is, 12.94 ± 3.45, N = 6, p = 0.89; Student's t test]. F–H, Representative traces of EJPs and mEJPs recorded in 0.4 mm Ca²⁺ at wild-type (F), cac^sfGFP-N (G), and cac^TagRFP-N NMJs (H). I, mEJP amplitude is unaffected in cac^sfGFP-N and cac^TagRFP-N (wild type, 0.91 ± 0.05, n = 9 NMJs from 4 larvae; cac^sfGFP-N, 0.99 ± 0.04, n = 13 NMJs from 4 larvae, p = 0.17, Student's t test; cac^TagRFP-N, 0.99 ± 0.04, n = 12 NMJs from 4 larvae, p = 0.20, Mann–Whitney U test). J, EJP amplitude is unchanged between wild-type, cac^sfGFP-N and cac^TagRFP-N NMJs (wild type, 30.87 ± 1.41, n = 9 NMJs from 4 larvae; cac^sfGFP-N, 29.64 ± 0.52, n = 13 NMJs from 4 larvae, p = 0.84, Mann–Whitney U test; cac^TagRFP-N, 26.70 ± 1.39, n = 12 NMJs from 4 larvae, p = 0.05, Student's t test). K, Quantal content is similar between wild-type, cac^sfGFP-N and cac^TagRFP-N NMJs (wild type, 34.8 ± 2.3, n = 9 NMJs from 4 larvae; cac^sfGFP-N, 30.6 ± 1.4, n = 13 NMJs from 4 larvae, p = 0.12, Student's t test; cac^TagRFP-N, 27.5 ± 1.7, n = 12 NMJs from 4 larvae, p = 0.03, Mann–Whitney U test). Not significant (ns) and *p < 0.05.