Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast and slow central synapses
- Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology - Graduate University, Japan
Figures
Figure 1 with 1 supplement
The endocytic blocker Dynasore or Pitstop-2 inhibits slow, fast-accelerating and fast endocytosis at the calyx of Held.
(A) Average traces of slow endocytic membrane capacitance changes (ΔCm) in response to a 5 ms depolarizing pulse (stepping from –70 mV to +10 mV) in the absence (control, black trace) or presence of …
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Figure 1—source data 1
The endocytic blocker Dynasore or Pitstop-2 inhibits slow, fast-accelerating and fast endocytosis at the calyx of Held.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Effect of intra-terminal loading of Dynamin-1 PRD peptide on endocytosis at the calyx of Held.
(A) Schematic illustration of presynaptic membrane capacitance (Cm) recording and loading of dynamin-1 proline-rich domain (Dyn1-PRD) peptide (sequence: PQVPSRPNRAP) into a calyceal terminal …
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Figure 1—figure supplement 1—source data 1
Effect of intra-terminal loading of Dynamin-1 PRD peptide on endocytosis at the calyx of Held.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 3 supplements
Endocytic blockers enhance activity dependent rapid synaptic depression, but do not prolong the recovery from depression at the calyx.
(A1, B1) A train of 30 EPSCs were evoked at the calyx of Held by afferent fiber stimulation at 10 Hz (A1) or 100 Hz (B1) in the absence (control, black traces) or presence of Dynasore (10–60 min, …
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Figure 2—source data 1
Endocytic blockers enhance activity dependent rapid synaptic depression, but do not prolong the recovery from depression at the calyx.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Effect of endocytic inhibitors or scaffold inhibitors on basal EPSC amplitude at the calyx and hippocampal CA1 synapses.
(A) Basal mean amplitude of EPSCs recorded from the calyx of Held in control (6.6±0.54 nA; n=22) was unaffected by Dynasore (7.6±0.67 nA; n=12; p=0.24), Pitstop-2 (6.6±0.61 nA; n=8; p=0.96), …
Figure 2—figure supplement 2
Postsynaptic AMPA receptor saturation does not contribute to enhancement of synaptic depression by endocytic blockers at the calyx of Held.
The low affinity glutamate receptor ligand kynurenic acid (Kyn, 1 mM) attenuated the EPSC amplitude (left top panel). In the presence of Kyn, the magnitude of enhancement of synaptic depression by …
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Figure 2—figure supplement 2—source data 1
Postsynaptic AMPA receptor saturation does not contribute to enhancement of synaptic depression by endocytic blockers at the calyx of Held.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
Endocytic blockers enhance STD but do not alter the recovery from STD time-course in 2.0 mM [Ca2+] irrespective of PT or RT at the calyx of Held.
Effects of Dynasore and Pitstop-2 on short-term synaptic depression (STD; A1, B1, and C1) and on the time course of normalized recovery from STD (A2, B2, and C2) during 100 Hz (A, B) or 200 Hz (C) …
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Figure 2—figure supplement 3—source data 1
Endocytic blockers enhance STD but do not alter the recovery from STD time-course in 2.0 mM [Ca2+] irrespective of PT or RT at the calyx of Held.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig2-figsupp3-data1-v1.xlsx
Figure 3
Scaffold machinery inhibitors have no effect on endocytic membrane retrievals at the calyx of Held.
The CDC42 inhibitor ML141 (10 µM, 10–60 min, cyan) or actin depolymerizer Latrunculin B (Lat-B, 10–60 min, 15 µM, red) had no effect on slow endocytosis in response to a 5 ms depolarizing pulse (A) …
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Figure 3—source data 1
Scaffold machinery inhibitors have no effect on endocytic membrane retrievals at the calyx of Held.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
Scaffold machinery inhibitors strongly enhance rapid synaptic depression activity independently, without prolonging the recovery from depression at the calyx.
(A1, B1) EPSCs (30x) evoked at the calyx of Held by afferent fiber stimulation at 10 Hz (A1) or 100 Hz (B1) in the absence (control, black) or presence of ML141 (10–60 min, cyan) or Lat-B (10–60 …
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Figure 4—source data 1
Scaffold machinery inhibitors strongly enhance rapid synaptic depression activity independently, without prolonging the recovery from depression at the calyx.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Effects of Latrunculin application protocols and [Ca2+] on STD.
(A) Two types of Latrunculin-A application protocols: perfusion (A1) and pre-incubation (A2). (B) In 2.0 mM [Ca2+], normalized amplitudes of EPSCs (30x at 100 Hz) in the presence (green circles) or …
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Figure 4—figure supplement 1—source data 1
Effects of Latrunculin application protocols and [Ca2+] on STD.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig4-figsupp1-data1-v1.xlsx
Figure 5
Endocytic and scaffold machineries co-operate for rapid vesicle replenishment during high-frequency transmission at the calyx of Held.
(A) Exponential curve fits to the time-course of synaptic depression during 100 Hz stimulation under control and in the presence of either endocytic blockers or scaffold cascade inhibitors. The …
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Figure 5—source data 1
Endocytic and scaffold machineries co-operate for rapid vesicle replenishment during high-frequency transmission at the calyx of Held.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
Endocytic blockers attenuate synaptic facilitation activity dependently at hippocampal CA1 synapses, but scaffold cascade inhibitors show no effect.
(A–D) A train of 30 EPSCs recorded from hippocampal CA1 pyramidal cells evoked by Schaffer collaterals stimulations at 10 Hz (A and C) or 25 Hz (B and D) in the absence (control, black) or presence …
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Figure 6—source data 1
Endocytic blockers attenuate synaptic facilitation activity dependently at hippocampal CA1 synapses, but scaffold cascade inhibitors show no effect.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Vacuolar (v-) ATPase blockers, Bafilomycin or Folimycin does not affect the short-term synaptic facilitation at the hippocampal CA1 synapse.
(A, B) EPSCs (30x) evoked in hippocampal CA1 pyramidal cells by Schaffer collateral stimulation at 10 Hz (A) or 25 Hz (B) in the absence (control, black) or presence of v-ATPase blockers Bafilomycin …
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Figure 6—figure supplement 1—source data 1
Vacuolar (v-) ATPase blockers, Bafilomycin or Folimycin does not affect the short-term synaptic facilitation at the hippocampal CA1 synapse.
- https://cdn.elifesciences.org/articles/90497/elife-90497-fig6-figsupp1-data1-v1.xlsx
Figure 7
Hypothetical vesicle replenishment scheme by endocytosis and scaffold-machineries during repetitive transmission at fast calyx and slow hippocampal CA1 synapses.
During high-frequency transmission, endocytosis driven site-clearance allows activity-dependent replenishment of new vesicles to release sites. This endocytic function counteracts synaptic …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | Dynasore | Abcam | Cat# ab120192 | 100 µM |
| Chemical compound, drug | Pitstop-2 | Abcam | Cat# ab120687 | 25 µM |
| Chemical compound, drug | ML141 | Abcam | Cat# ab145603 | 10 µM |
| Chemical compound, drug | Latrunculin A | Labchem -Wako, Fujifilm | 125–04363 | 20 µM |
| Chemical compound, drug | Latrunculin B | Abcam | Cat# ab144291 | 15 µM |
| Chemical compound, drug | Folimycin (or Concanamycin A) | Abcam | Cat# ab144227 | 67 nM |
| Chemical compound, drug | Bafilomycin A1 | Cayman Chemical | Cat# 11038 | 5 µM |
| Peptide, recombinant protein | Dynamin-1 PRD peptide | GenScript | (Custom ordered) | 1 mM Sequence: PQVPSRPNRAP |
| Chemical compound, drug | Strychnine HCl | Sigma-Aldrich | Cat# S8753 | 2 µM |
| Chemical compound, drug | Bicuculline Methiodide | Sigma-Aldrich | Cat# 14343 | 10 µM |
| Chemical compound, drug | D-AP5 | Tocris | Cat# 0106 | 50 µM |
| Chemical compound, drug | QX-314 bromide | Alomone Labs | Cat# Q-100 | 2 mM |
| Chemical compound, drug | DMSO, Sterile Filtered | Santa Cruz Chemicals | Cat# sc-359032 | See in Methods details |
| Other | EPC 10 USB Patch Clamp Amplifier | Heka Elektronik | N/A | See in Method details |
| Other | BX51WI upright microscope | Olympus | Cat# BX51WI | See in Method details |
| Other | VT1200S Vibratome | Leica | N/A | See in Method details |
| Other | Model 2100 Isolated Pulse Stimulator | A-M Systems | N/A | See in Method details |
| Other | TC-344C Dual Channel Temp Controller | Warner Instruments | TC-344C | See in Method details |
| Other | PatchStar Micromanipulator | Scientifica | PatchStar | For precise movement and positioning of recording probe and stimulation electrode |
| Other | Axiocam 506 mono camera system | Zeiss | Axiocam 506 mono | Visualization system for slice recording |
| Other | PIP 6 pipette puller | HEKA | PIP 6 | See in Method details |
| Other | Borosilicate glass capillary | King Precision Glass Inc. | N/A | (2.0 mm OD) |
| Software, algorithm | Patchmaster | HEKA | N/A | |
| Software, algorithm | ZEN 2 Lite | Zeiss | N/A | |
| Software, algorithm | IGOR Pro | Wavemetrics | N/A | |
| Software, algorithm | Microsoft Excel | Microsoft | N/A | |
| Software, algorithm | Corel Draw | Corel Corporation | N/A |
Additional files
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Supplementary file 1
Membrane capacitance and calcium current amplitudes with or without endocytic blockers or presynaptic scaffold inhibitors during slow, fast-accelerating, or fast endocytosis.
- https://cdn.elifesciences.org/articles/90497/elife-90497-supp1-v1.docx
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Supplementary file 2
Parameters of recovery from STD at 37°C and 1.3 mM Ca2+ with or without endocytic- or presynaptic scaffold-blockers.
- https://cdn.elifesciences.org/articles/90497/elife-90497-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90497/elife-90497-mdarchecklist1-v1.docx
