Volume 90, Issue 3, Pages 492-498 (May 2016) Fast, Temperature-Sensitive and Clathrin-Independent Endocytosis at Central Synapses  Igor Delvendahl, Nicholas P. Vyleta, Henrique von Gersdorff, Stefan Hallermann  Neuron  Volume 90, Issue 3, Pages 492-498 (May 2016) DOI: 10.1016/j.neuron.2016.03.013 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Ultrafast Single-AP-Evoked Endocytosis (A) Top: voltage command (Vcommand) used for AP-evoked capacitance recordings in cMFBs. The AP waveform was recorded in a previous experiment with axonal stimulation. An example of a resulting Ca2+ current (ICa) is depicted below. On the right, Vcommand and ICa are shown on an expanded timescale and half-durations are indicated. Middle: example single-AP-evoked Cm trace (average of 40 consecutive sweeps). Bottom: corresponding series and membrane resistance (Rs and Rm, respectively). (B) TeNT-LC effectively blocks synaptic vesicle exocytosis in cMFBs. Top: voltage protocol with 3-ms depolarization from −80 mV to 0 mV. Middle: pharmacologically isolated Ca2+ current immediately after break-in (control, black) and after 3:00 min of whole-cell recording (5 μM TeNT-LC, blue). Bottom: corresponding Cm traces. Right: average data of Ca2+ current amplitudes and Cm increase (ΔCm) elicited with 3-ms depolarizations for control (black) and 5 μM TeNT-LC (blue; n represents number of cMFBs). Data are represented as mean ± SEM. (C) Grand average of AP-evoked Cm responses (black, n represents number of cMFBs). Blocking synaptic vesicle exocytosis with 5 μM TeNT-LC (blue) revealed a transient Cm increase not related to exocytosis. Subtraction (gray) shows the time course of endocytosis following a single AP. See also Figure S1. Neuron 2016 90, 492-498DOI: (10.1016/j.neuron.2016.03.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Highly Temperature-Sensitive Endocytosis at Hippocampal and Cerebellar Mossy Fiber Synapses (A) Example traces of Cm recordings in hMFBs evoked by a train of ten stimuli (1-ms depolarizations to +20 mV) delivered at 50 Hz (arrow) at 36°C (red), 30°C (gray), and 24°C (blue). Lower traces represent corresponding membrane and series resistance (Rm and Rs, respectively). (B) Grand average Cm traces recorded at 36°C, 30°C, and 24°C (color code as in A; n represents number of hMFBs). The decay of the grand average traces was best fit with the sum of two exponentials with time constants of 1.1 s (67%) and 11.3 s for 36°C, the sum of two exponentials with time constants of 2.3 s (43%) and 7.4 s for 30°C, and a single exponential function with time constant of 10.7 s for 24°C. (C) Average amplitude-weighted time constant (τw) of the exponential fits to the Cm decay for the three temperatures. Data are represented as mean ± SEM, n is given in (B). (D) Left: histogram of Q10 values by bootstrap analysis of endocytosis time constants obtained in hMFBs based on τw data shown in (C). Right: corresponding histogram of Q10 values by bootstrap analysis of endocytosis time constants obtained in cMFBs using 3-ms depolarizations at 23°C and 36°C (cf. Figure S2A). See also Figure S2. Neuron 2016 90, 492-498DOI: (10.1016/j.neuron.2016.03.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Clathrin-Independent Single-AP-Evoked Endocytosis (A) Top: voltage command used for AP-evoked Cm recordings in cMFBs. Bottom: grand averages of AP-evoked Cm responses (n represents number of cMFBs) for control (black), application of a clathrin-inhibitor (pitstop 2, 25 μM; orange), a dynamin-inhibitor (dynasore, 100 μM; red), and an actin-inhibitor (latrunculin A, 25 μM; green). Gray solid lines are exponential fits to the Cm decay. (B) Average time constants of endocytosis and amplitudes of AP-evoked ΔCm with endocytosis inhibitors (color code as in A). The speed of endocytosis was unaltered by pitstop 2 (p = 0.92) but significantly slowed by dynasore and latrunculin A (p < 0.001). Data are represented as mean ± SEM. See also Figure S3. Neuron 2016 90, 492-498DOI: (10.1016/j.neuron.2016.03.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Distinct Molecular Modes of Endocytosis (A) Top: voltage command of 20 APs at a frequency of 300 Hz for Cm recordings in cMFBs. Bottom: corresponding Ca2+ currents with slight amplitude facilitation. (B) Example Cm traces following 20 APs at 300 Hz for control (black), application of a clathrin-inhibitor (pitstop 2, 25 μM; orange), a dynamin-inhibitor (dynasore, 100 μM; red), an actin-inhibitor (latrunculin A, 25 μM; green), and with TeNT-LC (5 μM, blue). (C) Top: 30-ms voltage step from −80 mV to 0 mV. Bottom: corresponding Ca2+ current. (D) Example Cm traces following a 30-ms depolarization as shown in (C). (E) Summary of the effect of endocytosis inhibitors on exocytosis evoked by different stimuli recorded in cMFBs (color code as in B and D). The endocytosis inhibitors dynasore, latrunculin A, and pitstop 2 had no effect on exocytosis evoked by weaker stimuli (1 AP, 20 APs, 1 ms, and 3 ms), whereas TeNT-LC completely blocked synaptic vesicle exocytosis. For 30-ms step pulses, exocytosis was reduced by latrunculin A. Inset: enlargement of the first three stimulus types on a logarithmic scale. (F) Summary of the effect of endocytosis inhibitors on time constants of endocytosis evoked by different stimuli. The clathrin inhibitor (pitstop 2, orange) had no effect on fast endocytosis evoked by single APs and AP trains (arrows) but stronger impact on endocytosis evoked by depolarizations of 1, 3, or 30 ms duration. In contrast, a dynamin-inhibitor (dynasore, red) and an actin-inhibitor (latrunculin A, green) reduced the speed of endocytosis evoked by all tested stimuli. Throughout the figure, data are represented as mean ± SEM and asterisks indicate significance level with Kruskal-Wallis tests and post hoc Mann-Whitney U tests both with Bonferroni-Holm correction. Asymmetric error bars for 1–30 ms depolarizations represent the inverse of the SEM boundaries of the rate constants (see Supplemental Experimental Procedures). See also Figures S3 and S4. Neuron 2016 90, 492-498DOI: (10.1016/j.neuron.2016.03.013) Copyright © 2016 Elsevier Inc. Terms and Conditions