Volume 123, Issue 3, Pages 521-533 (November 2005) A Slowed Classical Pathway Rather Than Kiss-and-Run Mediates Endocytosis at Synapses Lacking Synaptojanin and Endophilin  Dion K. Dickman, Jane Anne Horne, Ian A. Meinertzhagen, Thomas L. Schwarz  Cell  Volume 123, Issue 3, Pages 521-533 (November 2005) DOI: 10.1016/j.cell.2005.09.026 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Synaptic Vesicles Are Fewer but Clustered in Synaptic Terminals Mutant for synj (A and B) Neuromuscular varicosities from third-instar larvae, shown at the same magnification. By comparison with controls, synj varicosities (B) have fewer synaptic vesicles, which, at some locations (arrow) close to the T bar ribbon of an active zone, are of larger diameter. Scale bar: 1 µm. (C) Synaptic sites, as indicated by T bar ribbons (arrowheads), lie closer together in locations not typical of control terminals. (D) Enlarged view of mutant synapse showing residual large vesicle profiles (arrow) associated with an active-zone T bar ribbon. Scale bar (in [D] for [C] and [D]): 100 nm. (E) The packing densities (left) and spacings (Ra/Re; see Supplemental Data) of synaptic vesicles in neuromuscular varicosities indicate that the reduced populations of synaptic vesicles are more clustered in mutant terminals than in their corresponding controls. The values of Ra and Re were each significantly higher in the mutant (both, p < 0.005), and their ratios were 0.85 in synj and 1.1 in wild-type, a difference that was also significant (p < 0.05). The distribution of vesicle diameters in synj neuromuscular varicosities (right) is shifted to an increased representation of vesicles >∼36 nm in diameter (arrow). Above this size, the distributions of diameters for synj and wild-type varicosities differed by eye in frequency histograms (data not shown), as also revealed by the cumulative-frequency plot of all vesicle sizes. Error bars are mean ± SEM. Cell 2005 123, 521-533DOI: (10.1016/j.cell.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Depletion and Recovery of the Synaptic-Vesicle Pool at Synapses Lacking Synaptojanin or Endophilin All error bars are mean ± SEM. (A) Control, synj, and endo mutants were stimulated at 10 Hz for 10 min (until arrow) and subsequently allowed to recover while monitoring this recovery with stimulation at 0.2 Hz. EJP amplitudes were averaged (binning 2 s of response for each 10 Hz time point), normalized to prestimulus amplitudes, and plotted as a function of time. (B) Representative traces of experiments in (A), with typical individual EJPs shown on a faster timescale, below their corresponding time points. (C and D) Determination of the size of the functional synaptic-vesicle pool in control and synj mutant synapses. (C) shits1 and shits1;synjLY/Df(2R)x58-7 mutants were stimulated at 10 Hz at 32°C to deplete the functional synaptic-vesicle pool. EJP amplitudes were normalized and averaged as in (A). (D) The total quanta released during 10 Hz stimulation in (A) compared with the total functional synaptic-vesicle pool in (C). During 10 Hz stimulation, 347,000 ± 20,200 synaptic vesicles (mean ± SEM, n = 7) are released from control synapses, 99,600 ± 8,100, from synj (n = 11), and 79,200 ± 14,200 from endo (n = 7). In the absence of recycling, the releasable pool in shi was 49,900 ± 4,600 vesicles (n = 4), while in shi;synj it was 18,500 ± 2,200 (n = 4). Cell 2005 123, 521-533DOI: (10.1016/j.cell.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 mEJP Amplitude Changes after a Tetanus (A) Representative traces of mEJP recordings from control synapses in prestimulus time points (controlpre) and 10 min after the tetanus (controlpost+10). (B and C) Similar representative traces for synj (B) and endo (C). (D) Cumulative-probability histograms showing mEJP amplitude distributions in control synapses during prestimulus conditions (controlpre, n = 3158), immediately following the tetanus (controlpost, n = 418), and 10 min following the tetanus (controlpost+10, n = 1030). All curves are significantly different (Kolmogorov-Smirnov [KS] test, p < 0.001). (E) Similar histograms for synj during prestimulus conditions (synjpre, n = 1935) compared with controlpre (not significantly different; KS test, p > 0.05), synjpost (n = 823), and synjpost+10 (n = 429). Both synjpost and synjpost+10 distributions are significantly different from synjpre and controlpre (KS test, p < 0.001). (F) Similar histograms of endo mutant (endopre, n = 2275) distributions relative to controlpre and endopost+10 (n = 1492). All are significantly different from each other (KS test, p < 0.001). Events from 2 min of recordings from each of at least 5 animals were pooled for each genotype and condition. Cell 2005 123, 521-533DOI: (10.1016/j.cell.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Loading and Unloading FM1-43 Dye in Cycling and Re-Forming Synaptic Vesicles All error bars are mean ± SEM. (A–C) Synapses on muscles 6 and 7 loaded with FM1-43 in control, synj, and endo larvae. Animals were dissected and stimulated with 90 mM K+ and 2 mM Ca2+ for 10 min in the presence of FM1-43 dye to load recycling synaptic vesicles, then washed in 5 mM K+, 0 mM Ca2+ saline for 12 min, and finally imaged. Inset: individual labeled boutons of each genotype. (D) Control larvae failed to load dye when the nerve cord was cut, and the dye was instead applied in 5 mM K+, 2 mM Ca2+. (E–G) Synaptic terminals were unloaded following an additional 5 min incubation in 90 mM K+ without dye. (H) Loading was quantified (n = 7 for wt, n = 8 for synj, n = 7 for endo, and n = 5 for wt controls; see Experimental Procedures). (I–K) Control, synj, and endo synapses were loaded in a manner similar to that in (A)–(C) using endogenous activity from the nerve cord during 10 min incubation in 5 mM K+, 2 mM Ca2+ and dye. (L) Synapses with a severed nerve were not competent to load in the same conditions. (M–O) These loaded synapses were unloaded by 5 min incubation in 90 mM K+. (P) Loading was quantified (n = 5 for all genotypes). (Q–S) Depletion/re-formation loading in shi (shits1) (R) and shi;synj (shits1;synjLY/Df(2R)x58-7) (S) mutant terminals. Larvae were dissected and equilibrated to 34°C in 5 mM K+, 2 mM Ca2+ saline, then depleted of synaptic vesicles with a 5 min incubation in 90 mM K+, 2 mM Ca2+, and were finally allowed to re-form synaptic vesicles for 15 min at 22°C in 5 mM K+, 2 mM Ca2+ saline. FM1-43 was not loaded when larvae were treated similarly but the dye was removed before the 15 min recovery phase (Q). (T) Loading was quantified (n = 9 in shi, 11 in shi;synj, and 4 in shi controls). Scale bar: 10 µm for (A)–(G), 8 µm for (I)–(O), and 2 µm for (Q)–(S). Images were acquired with identical settings within each experimental group: (A–G), (I–O), (Q–S). All mutant loading was above background (p < 0.05). Cell 2005 123, 521-533DOI: (10.1016/j.cell.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Stimulation Frequency Determines Steady-State Transmission in Wild-Type, synj, and endo Synapses All error bars are mean ± SEM. (A) 1 Hz stimulation for 10 min in control (n = 4), synj (n = 6), and endo (n = 4) synapses. For each graph, 2 s of responses was averaged at each time point, normalized to initial EJP amplitudes, and plotted as a function of time. (B) A comparison of 5 and 10 Hz stimulation for 5 min in control (n = 4 and 5), synj (n = 7 and 4), and endo (n = 6 and 6) terminals. (C) Sequential stimulation at 1 Hz for 2 min, 7 Hz for 2 min, 10 Hz for 3 min, and 20 Hz for 3 min in control (n = 6) and synj (n = 7) terminals. (D) Sequential stimulation at 7 Hz for 3 min, 20 Hz for 3 min, 7 Hz for 5 min, and 0.2 Hz for 5 min in synj (n = 6) and endo (n = 5). Note that EJP amplitudes are dependent on the stimulation frequency and can recover from high-frequency trains, as seen in Figure 2, to reach the steady-state amplitude characteristic of that frequency. (E) Steady-state EJP amplitude at each frequency for wild-type, synj, and endo terminals. The data in (A)–(D) were fit with the equation f(t) = A1e−t/τ1 + A2e−t/τ2 + B to determine the normalized steady-state amplitude, B, at each frequency. (F) Quanta released per second during steady states at 1 Hz, 5 Hz, 7 Hz, 10 Hz, and 20 Hz in control, synj, and endo terminals. Quanta were calculated by dividing the EJP amplitudes at steady state by the mEJP amplitude and introducing a correction for nonlinear summation (Martin, 1955). Cell 2005 123, 521-533DOI: (10.1016/j.cell.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 A Slow Classical Endocytotic Pathway Can Explain the Endocytotic Defects in synj and endo Mutants Under conditions of low activity (left panels), a functional synaptic-vesicle pool and an EJP with normal amplitude can be maintained in both wild-type and mutant terminals. High-frequency stimulation increases the rate of exocytosis (center and right panels), but, in wild-type terminals, endocytotic rates increase to maintain a functional vesicle pool of nearly prestimulus size. In synj or endo terminals, however, endocytosis rates cannot adequately increase. While exocytotic rates are greater than endocytotic (center panel), the releasable pool is depleted, and the amplitude of the EJP declines. A new steady state is reached once the depletion of this pool is sufficient to reduce the rate of exocytosis to a level that is equal to the endocytotic rate (right panels). The thickness of the arrows represents the rates (vesicles/s) at which vesicles are cycled between intracellular and plasma-membrane pools. Cell 2005 123, 521-533DOI: (10.1016/j.cell.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions