Fast, Temperature-Sensitive and Clathrin-Independent Endocytosis at Central Synapses

Abstract

The fusion of neurotransmitter-filled vesicles during synaptic transmission is balanced by endocytotic membrane retrieval. Despite extensive research, the speed and mechanisms of synaptic vesicle endocytosis have remained controversial. Here, we establish low-noise time-resolved membrane capacitance measurements that allow monitoring changes in surface membrane area elicited by single action potentials and stronger stimuli with high-temporal resolution at physiological temperature in individual bona-fide mature central synapses. We show that single action potentials trigger very rapid endocytosis, retrieving presynaptic membrane with a time constant of 470 ms. This fast endocytosis is independent of clathrin but mediated by dynamin and actin. In contrast, stronger stimuli evoke a slower mode of endocytosis that is clathrin, dynamin, and actin dependent. Furthermore, the speed of endocytosis is highly temperature dependent with a Q10 of ∼3.5. These results demonstrate that distinct molecular modes of endocytosis with markedly different kinetics operate at central synapses.

Figure 1. Ultrafast single-AP-evoked endocytosis

(A) Top: Voltage command (V_command) 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 (I_Ca) is depicted below. On the right, V_command and I_Ca are shown on an expanded time scale and half-durations are indicated. Middle: Example single-AP-evoked C_m trace (average of 40 consecutive sweeps). Bottom: Corresponding series and membrane resistance (R_s and R_m, 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 minutes of whole-cell recording (5 μM TeNT-LC, blue). Bottom: Corresponding C_m traces. Right: Average data of Ca²⁺ current amplitudes and C_m increase (ΔC_m) 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 C_m responses (black, n represents number of cMFBs). Blocking synaptic vesicle exocytosis with 5 μM TeNT-LC (blue) revealed a transient C_m increase not related to exocytosis. Subtraction (gray) shows the time course of endocytosis following a single AP. See also Figure S1.

Figure 2. Highly temperature-sensitive endocytosis at hippocampal and cerebellar mossy fiber synapses

(A) Example traces of C_m 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 (R_m and R_s, respectively). (B) Grand average C_m 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 (τ_w) of the exponential fits to the C_m 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 rates obtained in hMFBs based on τ_w data shown in panel C. Right: Corresponding histogram of Q₁₀ values by bootstrap analysis of endocytosis rates obtained in cMFBs using 3-ms depolarizations at 23° C and 36° C (cf. Figure S2A). See also Figure S2.

Figure 3. Clathrin-independent single-AP-evoked endocytosis

(A) Top: Voltage command used for AP-evoked C_m recordings in cMFBs. Bottom: Grand averages of AP-evoked C_m 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 C_m decay. (B) Average time constants of endocytosis and amplitudes of AP-evoked ΔC_m 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.

Figure 4. Distinct molecular modes of endocytosis

(A) Top: Voltage command of 20 APs at a frequency of 300 Hz for C_m recordings in cMFBs. Bottom: Corresponding Ca²⁺ currents with slight amplitude facilitation. (B) Example C_m 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 C_m 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 panel 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. See also Figures S3 and S4.