Synaptotagmin-1 is the Ca2+ sensor for fast striatal dopamine release
- Department of Neurobiology, Harvard Medical School, United States
Figures
Figure 1 with 4 supplements
Synaptotagmin-1 is required for synchronous dopamine release.
(A) Schematic of the experimental setup for Cre-dependent expression of channelrhodopsin variants using mutant mice (B–D) and AAVs (F–I). (B–D) Sample traces (B, average of four sweeps), and …
Figure 1—figure supplement 1
Carbon fiber recordings.
(A) Sample traces and summary plot of the calibration of a carbon fiber electrode (CFE) with different concentrations of dopamine applied to the electrode by a puff pipette. The standard curve …
Figure 1—figure supplement 2
Synaptotagmin-1 is present in TH-positive striatal synaptosomes.
(A) Schematic of the striatal synaptosome preparation. (B, C) Sample confocal images (B) and quantification (C) of striatal synaptosomes stained for Synaptotagmin-1, the release site marker Bassoon …
Figure 1—figure supplement 3
Action potential firing of dopamine axons persists upon Synaptotagmin-1 knockout.
(A) Schematic of the extracellular recording. Dopamine axonal firing was induced optogenetically in the dorsal striatum and field potentials were recorded with an extracellular recording pipette …
Figure 1—figure supplement 4
Release evoked by electrical stimulation is abolished upon Synaptotagmin-1 knockout.
(A) Schematic of the experiment. Electrical stimulation activates dopamine fibers and cholinergic interneurons, and cholinergic innervation of dopamine axons accounts for as much as ~90% of the …
Figure 2
Dopamine axon structure and depolarization induced dopamine release are intact after ablation of Synaptotagmin-1.
(A) Sample 3D-SIM images of striatal brain sections of Syt-1 control and Syt-1 cKODA mice stained for the dopamine axon marker TH and the release site marker Bassoon. Volume rendered reconstructions …
Figure 3
Asynchronous dopamine release sustains extracellular dopamine in vivo after ablation of Synaptotagmin-1.
(A–E) Sample traces (A, average of four sweeps) and quantification of dopamine release (B–E) evoked by a 10 Hz stimulus train induced as described in Figure 1H before (black traces) and after …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | B6.SJL-Slc6a3tm1.1(cre)Bkmm/J; DATIRES-Cre | Bäckman et al., 2006 | JAX 006660, RRID:IMSR_JAX:006660 | |
| Genetic reagent (M. musculus) | B6.129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze; Ai32 | Madisen et al., 2012 | JAX 012569, RRID:IMSR_JAX:012569 | |
| Genetic reagent (M. musculus) | C57BL/6NTac-Syt1tm1a(EUCOMM)Wtsi/WtsiCnrm; Syt-1 floxed; Syt1lox/lox | Skarnes et al., 2011; Zhou et al., 2015; obtained from Dr. T.C. Südhof | EUCOMM (EM:06829), RRID:IMSR_EM:06829 | Conditional Synaptotagmin-1 floxed mice |
| Cell line (H. sapiens) | HEK293T | ATCC | Cat#: CRL-3216, RRID:CVCL_0063 | |
| Recombinant DNA reagent | AAV-flex-oChIEF-citrine | Addgene; Lin et al., 2009 | Plasmid# 50973, RRID:Addgene_50973 | |
| Chemical compound, drug | Nomifensine | Tocris | Cat. No. 1992 | |
| Chemical compound, drug | Tetrodotoxin | Tocris | Cat. No. 1078 | |
| Antibody | Mouse monoclonal IgG2a anti-Bassoon SAP7F407 (A85) | Enzo Life Sciences | Cat# ADI-VAM-PS003-F, RRID:AB_11181058 | IHC (1:500) |
| Antibody | Guinea pig polyclonal anti-Tyrosine hydroxylase (A111) | Synaptic Systems | Cat# 213 104, RRID:AB_2619897 | IHC (1:1000) |
| Antibody | Rabbit polyclonal anti-Synaptotagmin-1 antiserum (A24) | gift from Dr. T.C. Südhof | V216 | ICC (1:1000) |
| Other | Microdialysis probe | Harvard Apparatus | Item# CMA8309581 | |
| Other | Carbon fiber filaments | Goodfellow | Item# C 005722 | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | RRID:SCR_002285, https://imagej.net/Fiji/Downloads | Used for confocal synaptosome and 3D-SIM slice experiments |
| Software, algorithm | SoftWoRX | GE Healthcare | http://incelldownload.gehealthcare.com/bin/download_data/SoftWoRx/7.0.0/SoftWoRx.htm | Used for 3D reconstruction |
| Software, algorithm | Custom MATLAB code | Liu et al., 2018 | https://github.com/hmslcl/3D_SIM_analysis_HMS_Kaeser-lab_CL (Liu, 2017) | Used for analysis of 3D-SIM and synaptosome images |
| Software, algorithm | Prism8 | GraphPad | RRID:SCR_002798, https://www.graphpad.com/scientific-software/prism | Used for statistical analysis |
