Ciliary neuropeptidergic signaling dynamically regulates excitatory synapses in postnatal neocortical pyramidal neurons
- Department of Biology, Brandeis University, United States
Figures
Figure 1 with 1 supplement
Acute knockdown of ciliary proteins does not alter dendritic morphology of cortical pyramidal neurons.
(A) Representative images of a DIV11 pyramidal neuron expressing GFP (top), and an inhibitory neuron immunolabeled with GAD67 antibodies (bottom). Cilia (arrows) are immunolabeled using antibodies …
-
Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Gross neuronal morphology is unaltered upon acute knockdown of ciliary proteins.
(A and B) Relative fluorescence intensities of immunolabeled ciliary ARL13b (A) and cilia lengths (B) in neurons transfected with the indicated plasmids 48 hr post-transfection. Each dot is a …
-
Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
Acute knockdown of ciliary proteins increases the strength and number of excitatory synapses.
(A, B, and E) Representative images of pyramidal neuron dendrites immunolabeled with antibodies against GluA2 and VGlut1 (A and B), or Gephyrin and GAD65 (E). Cultures were transfected with GFP …
-
Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Presynaptic VGlut1 staining is increased upon acute knockdown of ciliary proteins.
(A and B) Relative fluorescence intensity of immunolabeled VGlut1 (A) or GAD65 (B) at GluA2/VGlut1 or Gephyrin/GAD65 colocalized puncta, respectively, in neurons transfected with the indicated …
-
Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig2-figsupp1-data1-v2.xlsx
Figure 3
Mean mEPSC amplitude in pyramidal neurons is increased following acute reduction of ciliary signaling.
(A) (Left) Representative mEPSC traces (top) and average waveforms (bottom) from neurons transfected with the indicated plasmids. (Right) mEPSC amplitude from neurons transfected with the indicated …
-
Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig3-data1-v2.xlsx
Figure 4
Disruption of ciliary signaling increases spontaneous firing without influencing intrinsic excitability.
(A) (Left) Representative voltage traces of spontaneous activity recorded from pyramidal neurons expressing GFP alone or shArl13b_1 and GFP. (Right) Average spontaneous firing rate for neurons …
-
Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
SSTR3 is localized to the cilia of excitatory, but not inhibitory, cortical neurons.
(A) Representative images of primary cilia immunolabeled with antibodies against AC3 and SSTR3 in GFP-expressing neurons in fixed cortical slices from P22 animals. Images at right show enlarged …
-
Figure 5—source data 1
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
SSTR3 is present in the majority of the cilia of NeuN-positive neurons and absent from multiple inhibitory neuron subtypes in the cortex.
(A) Representative images of NeuN, GAD67, and SSTR3 co-labeling in fixed and immunostained cortical tissue. Center and right panels are enlarged representative images from each indicated layer of …
-
Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 1 supplement
Pharmacological modulation of ciliary SSTR3 bidirectionally regulates excitatory synapses.
(A) Representative images of excitatory neuron dendrites immunolabeled with antibodies against Shank3 (Shk3) and VGlut1. Cultures were treated for 24 hr with 2 μM L-796,798 (SSTR3 agonist; purified …
-
Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Agonism and antagonism of SSTR3 alter excitatory synaptic strengths.
(A) Relative fluorescence intensity of VGlut1 at colocalized Shk3/VGlut1 puncta on neurons treated with 2 μM SSTR3 agonist L-796,798 or 1 μM MK-4256 SSTR3 antagonist. Cultures were immunostained at …
-
Figure 6—figure supplement 1—source data 1
Source data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/65427/elife-65427-fig6-figsupp1-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Rattus norvegicus) | Long–Evans | Charles River Laboratories | Strain:006, RRID:RGD_2308852 | |
| Transfected construct (Rattus norvegicus) | pAAV-hSyn-EGFP | Bryan Roth via Addgene | RRID:Addgene_50465 | |
| Antibody | αAC3 (Mouse monoclonal) | Encor | Cat#:MCA-1A12, RRID:AB_2744501 | IF(1:500) |
| Antibody | αAC3 (Rabbit polyclonal) | Encor | Cat#:RPCA-ACIII, RRID:AB_2572219 | IF(1:500) |
| Antibody | αARL13B (Mouse monoclonal) | NeuroMab | Cat#:N295B/66 (75-287), RRID:AB_234154 | IF(1:1000) |
| Antibody | αChAT (Goat polyclonal) | Millipore | Cat#:AB144P, RRID:AB_2079751 | IF(1:100) |
| Antibody | αGAD67 (Goat polyclonal) | R and D Systems | Cat#:AF2086, RRID:AB_2107724 | IF(1:2000) |
| Antibody | αGAD67 (Mouse monoclonal) | Millipore | Cat#:MAB5406, RRID:AB_2278725 | IF(1:2000) |
| Antibody | αGluA2 (Mouse monoclonal) | Gift from Gouaux lab, OHSU | IF(1:1000) | |
| Antibody | αIFT88 (Rabbit polyclonal) | ProteinTech | Cat#:13967–1-AP, RRID:AB_2121979 | IF(1:500) |
| Antibody | αPV (Mouse monoclonal) | Synaptic Systems | Cat#:195 011, RRID:AB_2619884 | IF(1:500) |
| Antibody | αShank3 (Guineapig polyclonal) | Synaptic Systems | Cat#:162 304, RRID:AB_2619863 | IF(1:1000) |
| Antibody | αSOM (Mouse monoclonal) | Thermo Fisher | Cat#:14-9751-82, RRID:AB_2572982 | IF(1:500) |
| Antibody | αSSTR3 (Rabbit polyclonal) | Biotrend-USA | Cat#:SS-830–50, RRID:AB_2196357 | IF(1:2000) |
| Antibody | αVGlut1 (Chicken polyclonal) | Synaptic Systems | Cat#:135 316, RRID:AB_2619822 | IF(1:1000) |
| Antibody | αVGlut1 (Guineapig polyclonal) | Synaptic Systems | Cat#:135 304, RRID:AB_887878 | IF(1:1000) |
| Antibody | αVIP (Guineapig polyclonal) | Synaptic Systems | Cat#:443 005, RRID:AB_2832228 | IF(1:500) |
| Recombinant DNA reagent | pLKO.1 (plasmid) | David Root via Addgene | RRID:Addgene_10878 | |
| Recombinant DNA reagent | pAAV-hSyn-EGFP (plasmid) | Bryan Roth via Addgene | RRID:Addgene_50465 | |
| Recombinant DNA reagent | pSUPER (plasmid) | OligoEngine | Cat#:VEC-PBS-0002 | |
| Recombinant DNA reagent | pSUPER-H1-shCep164 | This paper | pLRT18 | shRNA: 5’-CAACAACCACATCGAACTTA-3’ |
| Recombinant DNA reagent | pLKO-U6-shArl13b_1 | This paper | pLRT19 | shRNA: 5’-CCTGTCAGAAAGGTGACACTT-3’ |
| Recombinant DNA reagent | pSUPER-H1-shIft88 | This paper | pLRT26 | shRNA: 5’-CGAATGGCTTGGAGCTTATTA-3’ |
| Recombinant DNA reagent | pAAV-H1-shArl13b_2-hSyn-EGFP | This paper | pLRT67 | shRNA: 5’-GCTCAGGACATGATCTCATAA-3' |
| Commercial assay or kit | Zombie Green Fixable Viability Kit | BioLegend | Cat#:423111 | Cell viability assessment |
| Chemical compound, drug | L-796,778 | Gift from Merck Pharmaceuticals | SSTR3 selective agonist | |
| Chemical compound, drug | MK-4256 | MedChemExpress | Cat#:HY-13466 | SSTR3 selective antagonist |
| Chemical compound, drug | Propidium iodide | Thermo Fisher | Cat#:P3566 | |
| Software, algorithm | IGOR Pro | Wavemetrics | RRID:SCR_000325 | https://www.wavemetrics.com/products/igorpro/igorpro.htm |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | https://www.mathworks.com/products/matlab.html |
| Software, algorithm | Metamorph | Molecular Devices | RRID:SCR_002368 | http://www.moleculardevices.com/Products/Software/Meta-Imaging-Series/MetaMorph.html |
| Software, algorithm | R (version 4.0.3) | R | RRID:SCR_001905 | https://www.R-project.org/ |
| Software, algorithm | RStudio | RStudio | RRID:SCR_000432 | http://www.rstudio.com/ |
Additional files
-
Supplementary file 1
Statistical analyses used.
- https://cdn.elifesciences.org/articles/65427/elife-65427-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/65427/elife-65427-transrepform-v2.docx
