Synaptotagmin-1 is the Ca2+ sensor for fast striatal dopamine release

  1. Aditi Banerjee
  2. Jinoh Lee
  3. Paulina Nemcova
  4. Changliang Liu
  5. Pascal S Kaeser  Is a corresponding author
  1. Department of Neurobiology, Harvard Medical School, United States
3 figures, 1 table and 1 additional file

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 …

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 …

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
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)B6.SJL-Slc6a3tm1.1(cre)Bkmm/J; DATIRES-CreBäckman et al., 2006JAX 006660,
RRID:IMSR_JAX:006660
Genetic reagent (M. musculus)B6.129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze; Ai32Madisen et al., 2012JAX 012569,
RRID:IMSR_JAX:012569
Genetic reagent (M. musculus)C57BL/6NTac-Syt1tm1a(EUCOMM)Wtsi/WtsiCnrm; Syt-1 floxed; Syt1lox/loxSkarnes et al., 2011; Zhou et al., 2015; obtained from Dr. T.C. SüdhofEUCOMM (EM:06829),
RRID:IMSR_EM:06829
Conditional Synaptotagmin-1 floxed mice
Cell line (H. sapiens)HEK293TATCCCat#: CRL-3216, RRID:CVCL_0063
Recombinant DNA reagentAAV-flex-oChIEF-citrineAddgene; Lin et al., 2009Plasmid# 50973,
RRID:Addgene_50973
Chemical compound, drugNomifensineTocrisCat. No. 1992
Chemical compound, drugTetrodotoxinTocrisCat. No. 1078
AntibodyMouse monoclonal IgG2a anti-Bassoon SAP7F407 (A85)Enzo Life SciencesCat# ADI-VAM-PS003-F,
RRID:AB_11181058
IHC (1:500)
AntibodyGuinea pig polyclonal anti-Tyrosine hydroxylase (A111)Synaptic SystemsCat# 213 104,
RRID:AB_2619897
IHC (1:1000)
AntibodyRabbit polyclonal anti-Synaptotagmin-1 antiserum (A24)gift from Dr. T.C. SüdhofV216ICC (1:1000)
OtherMicrodialysis probeHarvard ApparatusItem# CMA8309581
OtherCarbon fiber filamentsGoodfellowItem#
C 005722
Software, algorithmFijiSchindelin et al., 2012RRID:SCR_002285, https://imagej.net/Fiji/DownloadsUsed for confocal synaptosome and 3D-SIM slice experiments
Software, algorithmSoftWoRXGE Healthcarehttp://incelldownload.gehealthcare.com/bin/download_data/SoftWoRx/7.0.0/SoftWoRx.htmUsed for 3D reconstruction
Software, algorithmCustom MATLAB codeLiu et al., 2018https://github.com/hmslcl/3D_SIM_analysis_HMS_Kaeser-lab_CL (Liu, 2017)Used for analysis of 3D-SIM and synaptosome images
Software, algorithmPrism8GraphPadRRID:SCR_002798, https://www.graphpad.com/scientific-software/prismUsed for statistical analysis

Additional files

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