ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal
- Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Sweden
- Department of Neuroscience, Karolinska Institutet, Sweden
Figures
Figure 1 with 1 supplement
Somatostatin and dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons distribution along the spinal cord by expansion and light-sheet microscopy.
(A) A schematic illustration of the lamprey spinal cord treated for expansion microscopy (ExM). The spinal cords were immunostained for somatostatin and tyrosine hydroxylase (TH) prior to the ExM …
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Figure 1—source data 1
Distribution of somatostatin and dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons along the spinal cord.
Quantification of the somatostatin and dopaminergic CSF-c neurons in four areas (areas 1–4) of spinal cord.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig1-data1-v1.xlsx
Figure 1—video 1
Three-dimensional (3D) light-sheet with expansion microscopy (ExLSM) explores spatial organization of somatostatin and dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons in the spinal cord.
Z-stack recorded with a light-sheet of an expanded sample of a lamprey spinal cord tissue. The reconstruction highlights the specific location of somatostatin (magenta) and dopamine (green) …
Figure 2
Somatostatin and dopamine in cerebrospinal fluid-contacting (CSF-c) neurons are stored in dense-core vesicles.
(A–C) Somatostatin (magenta) and α-tubulin (green) immunostaining in CSF-c neurons. Scale bar in A, 5 µm. (B, C) Selected Region of interest (ROIs) of somatostatin dense-core vesicles (DCVs) in the …
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Figure 2—source data 1
Somatostatin in cerebrospinal fluid-contacting (CSF-c) neurons store in dense-core vesicles (DCVs).
Analysis of somatostatin DCVs diameter measured with confocal and stimulated emission depletion (STED) microscopy.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig2-data1-v1.xlsx
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Figure 2—source data 2
Dopamine in cerebrospinal fluid-contacting (CSF-c) neurons store in dense-core vesicles (DCVs).
Analysis of dopamine DCVs diameter measured with confocal and stimulated emission depletion (STED) microscopy.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig2-data2-v1.xlsx
Figure 3 with 1 supplement
Acidic and alkaline pH decreased the number of somatostatin dense-core vesicles (DCVs) in the soma but did not affect gamma-Aminobutyric acid (GABA) intensity.
(A–F) Spinal cord slices in normal (pH 7.4), acidic (pH 6.5), and alkaline (pH 8.5) extracellular solution stained with an anti-somatostatin antibody (magenta). (A–C) Confocal and stimulated …
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Figure 3—source data 1
Effect of acidic or alkaline pH on somatostatin dense-core vesicles (DCVs) number density in cell area of somatostatin-expressing cerebrospinal fluid-contacting (CSF-c) neurons.
Quantification of somatostatin DCVs number density in cell area of somatostatin-expressing CSF-c neurons (µm–2) in the different pH.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig3-data1-v1.xlsx
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Figure 3—source data 2
Effect of acidic or alkaline pH on GABA signal in somatostatin-expressing cerebrospinal fluid-contacting (CSF-c) neurons.
Analysis of the GABA intensity signal in somatostatin-expressing CSF-c neurons in the different pH.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig3-data2-v1.xlsx
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Figure 3—source data 3
No correlation between GABA and somatostatin signals in somatostatin-expressing cerebrospinal fluid-contacting (CSF-c) neurons.
Analysis of GABA signal in and outside of somatostatin dense-core vesicles (DCVs) in soma and axon of somatostatin-expressing CSF-c neurons.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Effect of acidic or alkaline pH on somatostatin dense-core vesicles (DCVs) number density in cell volume of somatostatin-expressing cerebrospinal fluid-contacting (CSF-c) neurons.
(A–C) Representative volumetric imaging of somatostatin DCVs in the soma of the identified cell, shown as an axially colour-coded projection in the different conditions (n = 5, pH 7.4; n = 4, pH 6.5;…
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Figure 3—figure supplement 1—source data 1
Effect of acidic or alkaline pH on somatostatin dense-core vesicles (DCVs) number density in cell volume of somatostatin-expressing cerebrospinal fluid-contacting (CSF-c) neurons.
Quantification of somatostatin DCVs number density in cell volume (µm–3) of somatostatin-expressing CSF-c neurons in the different pH.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 2 supplements
Dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons did not respond to acidic and alkaline pH.
(A–C) Stimulated emission depletion (STED) images of dopamine-containing dense-core vesicles (DCVs) in the soma of CSF-c neurons in normal (pH 7.4), acidic (pH 6.5), and alkaline (pH 8.5) …
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Figure 4—source data 1
Effect of acidic or alkaline pH on dopamine dense-core vesicles (DCVs) number density in cell area of dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons.
Quantification of dopamine DCVs number density in cell area (µm−2) in dopaminergic CSF-c neurons in the different pH.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig4-data1-v1.xlsx
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Figure 4—source data 2
Effect of acidic and alkaline pH on action potential frequency in dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons.
Quantification of action potential frequency in dopaminergic CSF-c neurons at different pH conditions during 1 min.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Effect of acidic or alkaline pH on dopamine dense-core vesicles (DCVs) number density in cell volume of dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons.
(A–C) Representative volumetric imaging of dopamine DCVs in the soma of the identified cell, shown as an axially colour-coded projection in the different conditions (n = 3, pH 7.4; n = 3, pH 6.5; n …
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Figure 4—figure supplement 1—source data 1
Effect of acidic or alkaline pH on dopamine dense-core vesicles (DCVs) number density in the cell volume of dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons.
Quantification of dopamine DCVs number density in cell volume (µm−3) of dopaminergic CSF-c neurons in the different pH conditions.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig4-figsupp1-data1-v1.xlsx
Figure 4—figure supplement 2
Dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons are sensitive to fluid movement.
(A) A schematic illustration showing a dopaminergic CSF-c neuron (red) was patched and a pressure pipette was placed close to its bulb-like ending. (B) A short pressure pulse (20 p.s.i., 80 ms) …
Figure 5 with 3 supplements
Primary and motile cilia symmetry are present in the lamprey spinal cord.
(A, B) Confocal and stimulated emission depletion (STED) images of a cerebrospinal fluid-contacting (CSF-c) neuron cilium in a non-expanded spinal cord pre-stained with anti-α-tubulin antibodies. …
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Figure 5—source data 1
Quantification of a cilium diameter of cerebrospinal fluid-contacting (CSF-c) neuron with confocal microscopy in non-expanded spinal cord.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig5-data1-v1.xlsx
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Figure 5—source data 2
Quantification of a cilium diameter of cerebrospinal fluid-contacting (CSF-c) neuron with stimulated emission depletion (STED) microscopy in non-expanded spinal cord.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig5-data2-v1.xlsx
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Figure 5—source data 3
Quantification of a cilium diameter of cerebrospinal fluid-contacting (CSF-c) neuron with confocal microscopy in expanded spinal cord (expansion microscopy, ExM).
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig5-data3-v1.xlsx
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Figure 5—source data 4
Quantification of a cilia diameter of cerebrospinal fluid-contacting (CSF-c) neuron with stimulated emission depletion (STED) microscopy in expanded spinal cord (ExSTED).
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig5-data4-v1.xlsx
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Figure 5—source data 5
Quantification of a primary cilium diameter of cerebrospinal fluid-contacting (CSF-c) neuron with 9 + 0 symmetry with stimulated emission depletion (STED) microscopy in expanded spinal cord (ExSTED).
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig5-data5-v1.xlsx
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Figure 5—source data 6
Quantification of a motile cilium diameter of cerebrospinal fluid-contacting (CSF-c) neuron with 9 + 2 symmetry with stimulated emission depletion (STED) microscopy in expanded spinal cord (ExSTED).
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig5-data6-v1.xlsx
Figure 5—figure supplement 1
Three-dimensional (3D) expansion microscopy (ExM) of expanded cilia protruding to the central canal.
Representative volumetric imaging of expanded cilia (stained with α-tubulin) protruding into the central canal from cerebrospinal fluid-contacting (CSF-c) neurons, shown as an axially colour-coded …
Figure 5—figure supplement 2
Cilia diameter with different techniques: Box plot representation of cilia diameters, measured in the different conditions, confocal, stimulated emission depletion (STED), expansion microscopy (ExM), and ExSTED.
Averages and ± SD are 402 ± 25, 240 ± 20, 266 ± 17, and 218 ± 18 nm, respectively. Student’s paired t-test: ***p < 0.001 significant difference between cilia diameter in confocal and ExSTED (p = 2.8 …
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Figure 5—figure supplement 2—source data 1
Cilia diameters measured in cerebrospinal fluid-contacting (CSF-c) neurons with the different techniques: confocal, stimulated emission depletion (STED), expansion microscopy (ExM), and ExSTED microscopy.
- https://cdn.elifesciences.org/articles/73114/elife-73114-fig5-figsupp2-data1-v1.xlsx
Figure 5—video 1
Three-dimensional (3D) ExSTED visualizes cerebrospinal fluid-contacting (CSF-c) neurons and their cilia within the 3D geometry of the central canal.
Z-stack recorded with stimulated emission depletion (STED) of an expanded lamprey spinal cord tissue showing cilia (stained with α-tubulin) protrusion to central canal from CSF-c neurons with high …
Figure 6 with 1 supplement
Cilia symmetries in somatostatin and dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons.
(A) A schematic illustration of an expanded spinal cord stained for somatostatin (magenta), dopamine (blue), and α-tubulin (green). The gel was cut through the central canal and flipped 90° on the …
Figure 6—figure supplement 1
Polycystic kidney disease 2-like 1 (PKD2L1), acid-sensing ion channel 3 (ASIC3), and ARL13b expression on cerebrospinal fluid-contacting (CSF-c) neurons on mouse spinal cord.
(A–L) Two different CSF-c neurons showing immunoreactivity to the PKD2L1 channel (magenta) on their cilium stained with α-tubulin (green). Scale bar, A, G, 2 µm and B, H, 1 µm. (C–E) Selected ROI …
Figure 7 with 1 supplement
Cerebrospinal fluid-contacting (CSF-c) neurons have one or two cilia on their bulb protrusions.
(A) An expansion microscopy (ExM) image of an expanded somatostatin CSF-c neuron showing one cilium on its bulb (arrow). Scale bar, 5 µm. (B, C) A somatostatin CSF-c neuron with two cilia (arrow) (C)…
Figure 7—video 1
Cerebrospinal fluid-contacting (CSF-c) neurons might contain two cilia.
Z-stack recorded with confocal microscopy of an expanded lamprey spinal cord tissue (three-dimensional [3D] expansion microscopy [ExM]) showing a CSF-c neuron with two cilia on its bulb. Cilia were …
Figure 8
Somatostatin and dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons are two distinct cell types with contrasting function along the spinal cord.
(A) Schematic illustration of a cross-section of the lamprey spinal cord, with a somatostatin- and dopamine-expressing CSF-c neuron at the central canal and their axonal projections. (B) Summary of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Lampetra fluviatilis) | Spinal cord | Collected from the Ljusnan River, Hälsingland, Sweden | Freshly isolated from Lampetra fluviatilis | |
| Biological sample (Mus musculus) | Spinal cord | Janvier Labs, C57BL/6 | Freshly isolated from C57BL/6 | |
| Antibody | Anti-acetylated tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T6793, RRID:AB_477585 | IF (1:500) |
| Antibody | Anti-somatostatin (rat monoclonal) | Millipore | MAB354, RRID:AB_2255365 | IF (1:100) |
| Antibody | Anti-somatostatin 14-IgG (rabbit polyclonal) | Peninsula laboratories | Cat# T-4102.0400, RRID:AB_518613 | IF (1:1000) |
| Antibody | Anti-TH (mouse monoclonal) | Millipore | Cat# MAB318, RRID:AB_2201528 | IF (1:200) |
| Antibody | Anti-TH (rabbit polyclonal) | Millipore | Cat# AB152, RRID:AB_390204 | IF (1:500) |
| Antibody | Anti-dopamine (mouse monoclonal) | Millipore | Cat# MAB5300, RRID:AB_94817 | IF (1:400) |
| Antibody | Anti-GABA (mouse monoclonal) | Swant | Cat# Mab 3A12, RRID:AB_2314454 | IF (1:2000) |
| Antibody | Anti-polycystin-L (rabbit polyclonal) | Millipore | Cat# AB9084, RRID:AB_571091 | IF (1:500) |
| Antibody | Anti-ASIC3 (rabbit polyclonal) | Thermo Fisher Scientific | Cat# PA5-41022, RRID:AB_2576713 | IF (1:400) |
| Antibody | Anti-ARL13B (rabbit polyclonal) | Proteintech | Cat# 17711-1-AP, RRID:AB_2060867 | IF (1:500) |
| Antibody | Donkey-anti-rat-IgG-AF594 | Jackson ImmunoResearch Labs | Cat# 712-585-153, RRID:AB_2340689 | IF (1:500) |
| Antibody | Donkey-anti-rat-IgG-AF488 | Jackson ImmunoResearch Labs | Cat# 712-545-153, RRID:AB_2340684 | IF (1:200) |
| Antibody | Goat-anti-mouse-STAR635P | Abberrior | Cat# ST635P-1001-500 UG, RRID:AB_2893232 | IF (1:500) |
| Antibody | Goat-anti-rabbit-AF594 | Thermo Fisher Scientific | Cat# A-11037, RRID:AB_2534095 | IF (1:500) |
| Antibody | Donkey-anti-mouse-IgG-Cy3 | Jackson ImmunoResearch Labs | Cat# 715-165-150, RRID:AB_2340813 | IF (1:500) |
| Antibody | Donkey-anti-mouse-IgG-AF488 | Jackson ImmunoResearch Labs | Cat# 715-545-150, RRID:AB_2340846 | IF (1:200) |
| Other | NeuroTrace530/615 | Thermo Fisher Scientific | Cat# N21482, RRID:AB_2620170 | IF (1:1000) |
| Other | NeuroTrace640/660 | Thermo Fisher Scientific | Cat# N21483, RRID:AB_2572212 | IF (1:1000) |
| Other | Phalloidin-STAR635P | Abberior | IF (1:200) | |
| Peptide, recombinant protein | Neurobiotin | Vector Laboratories | Cat# SP-1120, RRID:AB_2313575 | Injection of 0.5% solution for intracellular labelling |
| Peptide, recombinant protein | Streptavidin-AF488 | Jackson ImmunoResearch | Cat# 016-540-084, RRID:AB_2337249 | IF (1:1000) |
| Chemical compound, drug | Glutamate receptor antagonist kynurenic acid | Tocris Ellisville, MO, USA | Bath perfusion, 2 mM | |
| Chemical compound, drug | GABAA receptor antagonist gabazine | Tocris Ellisville, MO, USA | Bath perfusion, 20 mM | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | RRID:SCR_002285 | |
| Software, algorithm | MATLAB | The Mathworks | RRID:SCR_001622 | |
| Software, algorithm | Impsector | Max-Planck Innovation | RRID:SCR_015249 | |
| Software, algorithm | Origin | OriginLab | RRID:SCR_014212 | |
| Software, algorithm | Imaris 9.1 | Bitplane | RRID:SCR_007370 | |
| Software, algorithm | Clampex and Clampfit | Molecular Devices, CA, USA | RRID:SCR_011323 | |
| Commercial assay or kit | Digoxigenin RNA Labeling kit | Roche Diagnostics | Catalog #11 277 073 910 | In situ hybridization |
| Commercial assay or kit | TSA Cy3 Plus Evaluation Kit | PerkinElmer | NEL763E001 | In situ hybridization |
| Antibody | Anti-DIG antibody coupled to HRP (sheep polyclonal) | Roche Diagnostics | RRID: AB_514497 | IF (1:2000) |
