Local axonal morphology guides the topography of interneuron myelination in mouse and human neocortex
- Erasmus MC University Medical Center, Netherlands
- Brown University, United States
Figures
Figure 1 with 3 supplements
Super-resolution microscopy of fast-spiking, PV+ interneuron axons.
(a) Experimental approach. Biocytin-filled fast-spiking PV+ interneurons from mPFC were analyzed using both confocal imaging and structured illumination microscopy (SIM) imaging. See also Figure …
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Figure 1—source data 1
Diameter measurements for axonal segments (f), branch order (g), and en passant boutons (h) of PV::WT cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Experimental flowchart for axonal reconstructions.
Figure 1—figure supplement 2
Axonal diameter analysis.
Interbranch axonal diameter was sampled at high spatial frequency from branch point to branch point. This representative example based on Figure 1e, depicts a 3rd order branch segment. (a) Axonal …
Figure 1—figure supplement 3
Locations of biocytin-filled and reconstructed PV+ and SOM+ cells.
(a) Maximum projection confocal image of a biocytin-filled mPFC SOM+ interneuron (red), with the distance to midline indicated. (b–e) Soma locations of Pvalb::cre,Ai14 cells (b), PV::UBE3A, PV::WT, …
Figure 2
PV+ interneuron axon diameter co-varies with myelination.
(a) Average SIM z-stack projection of a biocytin-filled PV+ interneuron axon (red) along with its myelination (MBP; green arrowhead), centered over a fourth branch order segment. Note that the …
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Figure 2—source data 1
Morphological measures in PV::WT cells: internode-to-branch point (d), branch order (e), and bivariate interbranch distance / axonal diameter values for myelinated and unmyelinated segments (f).
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig2-data1-v2.xlsx
Figure 3
APEX2 contrast-enhanced electron microscopy confirms that smaller-diameter axonal segments lack myelination.
(a) Experimental flowchart. Pvalb::cre mice were given unilateral injections into mPFC with AAV2/CAG-flex-mGFP-APEX2, and sacrificed for confocal and electron microscopy after 7 to 14 days. (b) …
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Figure 3—source data 1
Quantification of PV::WT cell axonal diameter by APEX2 contrast-enhanced electron microscopy (g).
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig3-data1-v2.xlsx
Figure 4 with 4 supplements
PV::TSC1 and PV::UBE3A mice exhibit reciprocal alterations of PV+ cell morphology.
(a) Overview of mouse breeding scheme. (b) Maximum projection confocal image of PV+ somata (red) in PV::UBE3A mice lacking UBE3A (green; top), and in PV::TSC1 mice showing high pS6 expression …
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Figure 4—source data 1
Soma area (c) and axon onset diameter (f) for PV::UBE3A, PV:WT, and PV::TSC1 cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
PV::TSC1 mice exhibit PV-specific deletion of Tsc1.
(a) Pvalb::cre mice were crossed with floxed Tsc1f/f to obtain heterozygous Pvalb::cre x homozygous Tsc1-/- mice. In the absence of Tsc1, mTOR signaling is disinhibited leading to excessive S6K …
Figure 4—figure supplement 2
PV::UBE3A mice exhibit PV+ cell-specific deletion of Ube3a.
(a) PV-specific knockout of Ube3a. (b) Low magnification confocal microscopy image showing immunofluorescence of Ube3a (green) and PV (red) in adult mPFC. Note that the remaining non-PV neuronal …
Figure 4—figure supplement 3
PV cell-specific mutations of Ube3a or Tsc1 do not alter PV+ cell density in mPFC.
(a) Confocal microscopy image showing immunofluorescence of PV (red) and DAPI (white) in adult mPFC for PV::UBE3A, PV::WT, and PV::TSC1. Scale bar, 100 µm. (b) Quantification of PV+ cell density. …
Figure 4—figure supplement 4
Electrophysiological properties of PV::TSC1, PV::WT, and PV::UBE3A cells.
(a) input resistance. p=0.741. (b) resting membrane potential. p=0.267. (c) Frequency-current plot, in response to square-wave current steps. p=0.999. PV::WT, n = 14 cells; PV::TSC1, n = 22 cells; …
Figure 5 with 1 supplement
PV::UBE3A and PV::TSC1 mice exhibit bi-directional alterations in PV+ interneuron axonal myelination.
(a) Experimental approach. Biocytin-filled fast-spiking PV+ interneurons from each genotype were first analyzed using confocal imaging. (b–d) Maximum projection image of a representative …
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Figure 5—source data 1
Total recovered myelination length (j), internode length (k), internode number (l), branch order (m), myelin onset distance (n), as well as bivariate interbranch distance / axonal diameter values for myelinated and unmyelinated segments of PV::UBE3A (p) and PV::TSC1 (q) cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Intact global myelination and mature oligodendrocyte density in mPFC of mice with PV cell-specific mutations of Ube3a or Tsc1.
(a) Confocal microscopy image showing immunofluorescence of MBP (green) and DAPI (white) for PV::UBE3A, PV::WT, and PV::TSC1. Scale bar, 100 µm. (b) Quantification of MBP+ area. p=0.256, …
Figure 6
mPFC SOM+ interneurons have subthreshold axonal morphology and are correspondingly unmyelinated.
(a) Experimental approach. Biocytin-filled regular-spiking SOM+ interneurons from mPFC were analyzed using both confocal imaging and SIM imaging. See also Figure 1—figure supplements 1–3. (b) …
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Figure 6—source data 1
Diameter measurements for axonal segments (f), branch order (g), and bivariate interbranch distance / axonal diameter values for myelinated and unmyelinated segments (h) of SOM::WT cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig6-data1-v2.xlsx
Figure 7 with 3 supplements
SOM::TSC1 cells are frequently myelinated.
(a) Overview of mouse breeding scheme. (b) SOM::TSC1 cells have enlarged somata. Left: Representative confocal images of SOM+ cells (red) confirm the increased expression of pS6 (green) in SOM::TSC1 …
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Figure 7—source data 1
Soma area (b), axon onset diameter (d), total recovered myelination length (i), internode number (j), internode length (k), myelin onset distance (n), as well as bivariate interbranch distance / axonal diameter values for myelinated and unmyelinated segments of SOM::TSC1 (p) cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
SOM::TSC1 mice exhibit SOM-specific deletions of Tsc1.
(a) Sst::cre mice were crossed with floxed Tsc1f/f to obtain heterozygous Sst::cre x homozygous Tsc1-/- mice (SOM::TSC1). In the absence of Tsc1, mTOR signaling is disinhibited leading to excessive …
Figure 7—figure supplement 2
SOM::TSC1 mice have a normal SOM+ cell density in mPFC.
(a) Confocal microscopy image showing immunofluorescence of SOM (red) and DAPI (white) in adult mPFC for SOM::WT, and SOM::TSC1. Scale bar, 100 µm. (b) SOM+ cell density in mPFC of SOM::TSC1 and …
Figure 7—figure supplement 3
Electrophysiological properties of SOM::TSC1 and SOM::WT cells.
(a) input resistance. p=0.018. (b) sag potential. p=0.023. (c) resting membrane potential. p=0.993. (d) AP threshold. p=0.605. (e) AP amplitude. p=0.994. (f) fast afterhyperpolarization (fAHP) …
Figure 8
Extensive axonal myelination of SOM::TSC1 cells.
(a) Experimental flowchart. Adult SOM::WT and SOM::TSC1 mice were injected with low-titer AAV9 cre-dependent GFP reporter virus in mPFC, and sacrificed 14 days later for MBP immunofluorescence …
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Figure 8—source data 1
MBP+ area (e) and CC1+ cell counts (g) in SOM::WT and SOM::TSC1 cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig8-data1-v2.xlsx
Figure 9
Myelination thresholds extend to fast-spiking interneurons in human cortex.
(a) Experimental approach. Biocytin-filled interneurons from human ex vivo resected cortical tissue were analyzed using confocal imaging. (b) Maximum projection confocal image of a representative …
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Figure 9—source data 1
Morphological measures in human fast-spiking neocortical interneurons: internode-to-branch point (h), and bivariate interbranch distance / axonal diameter values for myelinated and unmyelinated segments (j).
- https://cdn.elifesciences.org/articles/48615/elife-48615-fig9-data1-v2.xlsx
Videos
Video 1
Adult PV::TSC1 mice are ataxic.
Left: PV::WT littermate. Right: PV::TSC1 mouse showing ataxia.
Additional files
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Source code 1
Fiji source code for automated quantification of axonal diameter within user-defined segments based on the Gaussian full-width at half-maximum of the orthogonal cross-section of fluorescence intensity.
- https://cdn.elifesciences.org/articles/48615/elife-48615-code1-v2.ijm
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Supplementary file 1
Electrophysiological properties of Pvalb::cre,Ai14 PV+ cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-supp1-v2.docx
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Supplementary file 2
Electrophysiological properties of Sst::cre,Ai14 SOM+ cells.
- https://cdn.elifesciences.org/articles/48615/elife-48615-supp2-v2.docx
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Supplementary file 3
Electrophysiological properties of human fast-spiking interneurons.
- https://cdn.elifesciences.org/articles/48615/elife-48615-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/48615/elife-48615-transrepform-v2.pdf
