Article Figures & Data
Figures
- Figure 1.
LTD at the Drosophila NMJ. A, Representative recordings of control and LTD induction. EJCs were recorded at the NMJ while the segmental nerves innervating the corresponding muscle fibers were stimulated at the baseline frequency of 0.05 Hz. For induction of LTD, tetanus of 30 Hz (20 s) was delivered at ∼5 min after continuous recording. Calibration: 20 nA, 2 min. B, Summary of normalized EJC amplitude in control (n = 6) and LTD (n = 13) recordings. Time of −5 min refers to the time before the tetanus, which applies to all figures. Arrow indicates delivery of tetanus. C, Comparison of quantal content in the control and LTD recordings. Note that, in contrast to EJCs, the quantal content in the control remained stable within 35 min. D, Dependency of LTD on tetanus frequency (5–50 Hz, 20 s; 0.4 mm Ca2+). n = 4, 4, 4, 4, 6, 4, 4 for the tetanus frequency of 0, 5, 10, 20, 30, 40, 50 respectively. E, Dependency of LTD on Ca2+ concentrations (0.2–1 mm, 30 Hz for 20 s). n = 4, 4, 4, 4, 6, 4, 4 for no tetanus and 0, 0.2, 0.4, 0.7, 1.0 mm Ca2+, respectively. For Figures 1, 2, and 4 –7, all recordings were performed on M12 in segment 3–5 and at 0.35–0.4 mm Ca2+.
- Figure 2.
An experiment indicating normal NMJ function after the induction of LTD. Tetanus of 15 Hz (15 s) was delivered at 20 min after LTD induction (30 Hz, 20 s); it induced short-term facilitation, posttetanic potentiation, and partial reversal of LTD. A, Representative traces. Calibration: 20 nA, 2 min. B, Summary (n = 5). Arrows indicate the delivery of 30 and 15 Hz tetanus.
- Figure 3.
Induction of LTD at various longitudinal muscle fibers M4, M6, and M12 (A, B) and at different axons (C–E). A, Representative traces. Calibration: 20 nA, 2 min. B, Summary of normalized EJC amplitude. n = 4, 6, 4 for M4, M6, M12, respectively. C, A representative recording on M6 by retrograde stimulation of axon 2 on the nerve branch to M12. Calibration: 10 nA, 2 min. D, Summary of experiments on stimulating axon 2. n = 4. E, Comparison of the responses on M6 evoked by stimulating axon 2 and the total EJC evoked by stimulating the segmental nerve. Note that the total EJC amplitudes of the segmental nerve were larger than those of axon 2 after induction of LTD, suggesting that both axons were responsive after LTD induction. n = 4 for either group.
- Figure 4.
Analysis of mEJCs and local glutamate perfusion-induced currents. Changes of mEJCs in the control (A, C, E) and before and after LTD induction (B, D, E). A, B, Representative mEJC traces. C, D, Histogram and Gaussian fitting of mEJCs. E, Summary of mEJC frequency changes. Quantal size and mEJC frequency were gradually reduced in the control (A, C, E). After LTD induction (15 min), quantal size remained unchanged, but there was an increased number of larger events that form a second peak, suggesting two quantal releases (D). As a result, the total frequency of mEJC also increased after the tetanus compared with the control (E). Calibration: 1 nA, 0.5 s. n = 8 for either group. F, Locally applied glutamate (100 mm)-induced currents (“G”) did not significantly change after the induction of LTD. Top, Representative traces. Arrowheads indicate the glutamate-induced currents (“G”), which have slow kinetics; all other fast spikes are EJCs (“E”). Calibration: 20 nA, 1 min. Expanded traces before and after the 30 Hz tetanus are shown in the boxes (scale bars, 100 ms). Bottom, Summary. n = 6.
- Figure 5.
LTD was primarily reduced in the akt mutant alleles akt4226 and akt4226/akt1 (A) but not affected in the mutants of rutabaga, dunce, and latheo (B). A, Representative recordings (top) (calibration: 20 nA, 2 min) and summary of normalized EJC amplitude (bottom) in control and akt mutant alleles. WT, Wild type. B, Representative LTD recordings in the mutants of rutabaga (rut1), dunce (dunce1), and latheo (latheoP1).
- Figure 6.
The akt mutant (akt4226) exhibited similarly impaired LTD at lower Ca2+ concentration (0.2 mm Ca2+, at which LTD was the most pronounced in wild-type larvae) or by increased stimulation frequency (40 Hz). A, B, Reduction of LTD in the akt mutant at 0.2 mm Ca2+ was similar to LTD at 0.4 mm Ca2+ (refer to Fig. 1E). A, Representative recordings for wild type (WT) and akt4226. Calibration: 20 nA, 2 min. B, Summary of four recordings. C, D, LTD induced by 40 Hz tetanus was also primarily impaired in the akt mutant. C, Representative traces. Calibration: 20 nA, 2 min. D, Summary of four recordings.
- Figure 7.
Rescue of LTD in akt1 and akt4226/akt1 by acutely induced expression of hsp70-akt transgene. The hsp70-akt;akt1 embryos and larvae were given developmental daily heat shock exposures to overcome the lethality (see Materials and Methods). A, Rescue of LTD in hsp70-akt;akt1 after brief heat shock exposures. hsp70-akt;akt1 larvae were shifted to 18°C for 24–48 h after the last daily heat shock treatment (hsp70-akt;akt1, 18°C). These larvae showed impaired LTD like the akt mutants (p > 0.05 compared with akt4226 and akt4226/akt1; two-way ANOVA). For acutely inducing hsp70-akt expression, a group of larvae was brought from 18°C to RT for 3–6 h and then were given heat shocks (18°C→HS; two heat shock exposures of 15 min at 37°C, 2 h interval). n = 14 and 9 for the groups of 18°C and 18°C→HS, respectively. B, Rescue of LTD by acutely induced hsp70-akt expression in the heterozygous allele akt4226/akt1. The allele hsp70-akt;akt4226/akt1 at RT showed impaired LTD like the akt mutant (hsp70-akt;akt4226/akt1; RT). The defective LTD was rescued by three to four brief heat shock exposures (37°C, 15 min, 1.5–2 h intervals) (hsp70-akt;akt4226/akt1, RT→3–4 HS). n = 4 and 6 for the groups RT and RT-HS, respectively. Calibration: 20 nA, 2 min. C, D, Rescue of LTD appears to correlate with the amount of hsp70-akt expression. C, In hsp70-akt;akt1 larvae, LTD was partially rescued at RT and fully rescued after additional heat shock exposures (2 times for 15 min, 37°C, 2 h interval). n = 4 and 5 for the RT and RT-HS groups, respectively. D, In hsp70-akt;akt4226/akt1, impaired LTD was partially rescued after two heat shock exposures (15 min, 37°C, 2 h interval; n = 3) but was fully rescued after four heat shock exposures (see Fig. 6B; n = 3). n = 4 for the RT group.
- Figure 8.
Normal synaptic transmission and short-term plasticity (E–G) in the akt mutants akt4226 and akt4226/akt1. A, Representative traces of spontaneous mEJCs in wild type (WT) and the akt mutants. Calibration: 1 nA, 1 s. B, Summary of amplitude and frequency of mEJCs. n = 8 and 6 for WT and akt groups, respectively. C, Representative traces of EJCs at different Ca2+ concentrations (0.1, 0.2, and 0.4 mm) in wild type and the akt mutants. Calibration: for 0.1 and 0.2 mm Ca2+, 10 nA, 10 ms; for 0.4 mm Ca2+, 20 nA, 10 ms. D, Ca2+ dependency of EJCs: logarithmic plot of the power relationship in the range of 0.1–0.4 mm Ca2+. n = 6, 6, and 4 for the control (diamond), akt4226 (square), and akt4226/akt1 (triangle) groups, respectively. E, Normal STF during a short train of repetitive stimulation (25 Hz) in the akt mutants. Top, Representative traces. Bottom, Summary of normalized EJC amplitude. n = 5 for each group. F, Normal dependence of STF on stimulation frequency in the akt mutants. Trains of 20 stimuli were delivered at the frequency of 0.5–20 Hz. The amplitudes of the last 10 responses (EJCs) in each train were averaged and normalized to the average EJC amplitude at 0.5 Hz. Top, EJC traces representative of the average of last 10 EJCs for 0.5 and 10 Hz. Calibration: 2 nA, 10 ms. Bottom, Summary. n = 5 for each group. G, Normal posttetanic potentiation in the akt mutants. Continuous recordings were made at 0.2 Hz stimulation before and after the 10 Hz tetanus. Top, Representative traces. Calibration: 5 nA, 1 min. Bottom, Summary of normalized EJC amplitudes. n = 11, 10, and 6 for control, akt4226, and akt4226/akt1 groups, respectively. [Ca2+], 0.15 mm for E–G.
- Figure 9.
Expression of Akt at the Drosophila NMJ and reduced Akt expression in the hypomorphic akt mutant akt4226. NMJs were stained using a polyclonal anti-dAkt antibody and FITC-conjugated secondary antibody. Shown in the top are representative fluorescence images of Akt immunostaining at the NMJ of wild type (WT) and akt4226. Under the color images are inverted images of the corresponding fluorescence images (see Materials and Methods). Comparison of staining intensity between wild type and akt4226 is shown in enlarged images of synaptic boutons and nerve branches (corresponding to the boxes in the fluorescence images and inverted images). Note that the difference in staining intensity between wild type and the mutant appears to be more apparent at the nerve branches. n = 4 and 3 for WT and akt4226, respectively. Scale bars, 20 μm.
