1. Neuroscience

Ankyrin-R regulates fast-spiking interneuron excitability through perineuronal nets and Kv3.1b K+ channels

  1. Sharon R Stevens
  2. Colleen M Longley
  3. Yuki Ogawa
  4. Lindsay H Teliska
  5. Anithachristy S Arumanayagam
  6. Supna Nair
  7. Juan A Oses-Prieto
  8. Alma L Burlingame
  9. Matthew D Cykowski
  10. Mingshan Xue
  11. Matthew N Rasband  Is a corresponding author
  1. Department of Neuroscience, Baylor College of Medicine, United States
  2. Program in Developmental Biology, Baylor College of Medicine, United States
  3. The Cain Foundation Laboratories, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, United States
  4. Department of Pathology and Genomic Medicine, Houston Methodist Hospital, United States
  5. Department of Pharmaceutical Chemistry, University of California San Francisco, United States
  6. Department of Molecular and Human Genetics, Baylor College of Medicine, United States
https://doi.org/10.7554/eLife.66491
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Cite this article
  1. Sharon R Stevens
  2. Colleen M Longley
  3. Yuki Ogawa
  4. Lindsay H Teliska
  5. Anithachristy S Arumanayagam
  6. Supna Nair
  7. Juan A Oses-Prieto
  8. Alma L Burlingame
  9. Matthew D Cykowski
  10. Mingshan Xue
  11. Matthew N Rasband
(2021)
Ankyrin-R regulates fast-spiking interneuron excitability through perineuronal nets and Kv3.1b K+ channels
eLife 10:e66491.
https://doi.org/10.7554/eLife.66491
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8 figures, 2 videos, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
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AnkR is expressed in select neurons in the cortex and hippocampus.

(A) Immunostaining of coronal mouse brain for AnkR. Arrowhead indicates a red blood cell. Scalebars, left, 250 μm, right, 20 µm. (B) Immunoblot of P1, P15, P30, and 6-month C57BL/6 brain homogenates …

Figure 1—source data 1

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Figure 1—figure supplement 1
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AnkR immunostaining in control and Ank1 conditional knockout mice.

(A) Immunostaining of cortex (top) and hippocampus (bottom) for AnkR (red), Parvalbumin (Pv, green), and Somatostatin (SST, blue). Outlined arrowheads illustrate AnkR+Pv+ cells, solid arrowheads …

Figure 2 with 1 supplement
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AnkR interacting proteins.

(A) Immunostaining of mouse cortex using antibodies against AnkR (red) and β1 spectrin (green). Scalebar, 20 µm. (B) Immunoblot of β1 spectrin, AnkR, and IgG immunoprecipitation reactions using …

Figure 2—source data 1

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Figure 2—figure supplement 1
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AnkR interacting proteins.

(A, B) Immunostaining of cortex (A) and hippocampus (B) in control and AnkR-deficient mouse brain using antibodies against AnkR (red), β1 spectrin (green), and the nuclear marker Hoechst. Scale …

Figure 3 with 2 supplements
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AnkR maintains perineuronal net density and structure.

(A) Fluorescent WFA labeling of PNNs in coronal sections of cortex and hippocampus in one-month-old mice. Genotypes of the respective mice are indicated. Boxed regions are shown to the right. …

Figure 3—source data 1

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Figure 3—figure supplement 1
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Labeling of control and AnkR cKO neurons in cortex using fluorescent WFA and antibodies against AnkR.

Scalebar, 50 μm.

Figure 3—figure supplement 2
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WFA labeling is reduced across cortical regions.

(A, B) Corrected Total Cell Fluorescence (CTCF) across cortical regions at one- and 12 months of age. RSP, retrosplenial cortex; MOp, primary motor cortex; MOs, secondary motor cortex; SSp, primary …

Figure 3—figure supplement 2—source data 1

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Figure 4 with 1 supplement
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Mice lacking AnkR in GABAergic forebrain interneurons have reduced anxiety.

(A) Thirty min-long recordings of mouse trajectories in the open-field assay. Genotypes are indicated. (B) The percent of time spent in the center or perimeter of the open field. (C) The percent of …

Figure 4—figure supplement 1
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Quantification of open field and elevated plus maze assays.

(A–C) Measurements of velocity (A), distance traveled (B), and rotations (C) in the open field test. (D, E) Measurements of velocity (D) and distance (E) in the elevated plus maze assay. In all …

Figure 5 with 2 supplements
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WFA+ neurons in Ank1F/F;Dlx5/6-Cre mice have abnormal action potentials and spike train characteristics.

(A) A representative action potential illustrating the measurement of action potential parameters. AHP, afterhyperpolarization. (B–D) Summary data showing the action potential latency (B), action …

Figure 5—figure supplement 1
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Flourescein-WFA labeling of perineuronal nets in live slices for electrophysiology.

(A) Top: low-magnification image of a coronal slice from the left hemisphere of an Ank1f/f mouse after recording. Lines on the brain slice are the indentations resulted from the mesh used to hold …

Figure 5—figure supplement 1—source data 1

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Figure 5—figure supplement 2
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Analysis of miniature excitatory postsynaptic currents.

(A) Representative traces of miniature excitatory postsynaptic currents (mEPSCs) recorded at –70 mV (top) and miniature inhibitory postsynaptic currents (mIPSCs) recorded at +10 mV (bottom) from Ank1…

Figure 5—figure supplement 2—source data 1

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Figure 6 with 1 supplement
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AnkR binds to Kv3.1b K+ channels and is both necessary and sufficient for its membrane localization and clustering.

(A, B) Immunostaining of 1-month-old somatosensory cortex for AnkR (red) and Kv3.1b (green). Low-magnification images are shown in (A) and high-magnification images in (B). The genotypes analyzed …

Figure 6—figure supplement 1
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Kv3.1b membrane localization requires AnkR.

(A) Immunolabeling of 12-month-old somatosensory cortex from Ank1F/F and Ank1F/F;Dlx5/6-Cre mice using antibodies against AnkR (red) and Kv3.1b (green). Scalebar, 50 μm. (B) Immunoblots of brain …

Figure 6—figure supplement 1—source data 1

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Figure 7
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AnkR is highly expressed in the perisomatic region of Pv+ interneurons which are surrounded by PNNs, a specialized ECM structure.

AnkR is a scaffolding protein that binds to and stabilizes PNN-associated CAMs (including NrCAM and PlexinA4) and ion channels (including Kv3.1b) by linking them to the β1-α2 spectrin-based …

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Videos

Video 1
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WFA in 12-month Ank1F/F.

Scalebar, 5 µm.

Video 2
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WFA in 12-month Ank1F/F;Dlx5/6-Cre.

Scalebar, 5 µm.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
gene (human)ANK1NCBI.NLM.NIH.gov/geneGene ID: 286
HGNC:HGNC:492
gene (Mus musculus)Ank1NCBI.NLM.NIH.gov/geneGene ID: 11733
MGI:MGI:88024
Strain, strain background (Mus musculus, male and female)C57BL/6J,
Wild type, WT		The Jackson LaboratoryJAX:000664See Materials and methods, Section Animals
Strain, strain background (Mus musculus, female)Nestin-Cre,
Nes-Cre		The Jackson LaboratoryJAX:003771See Materials and methods, Section Animals
Strain, strain background (Mus musculus, male)Dlx5/6-CreThe Jackson LaboratoryJAX:008199See Materials and methods, Section Animals
Strain, strain background (Mus musculus, male)Chat-Cre
ChAT-Cre		The Jackson LaboratoryJAX:006410See Materials and methods, Section Animals
Strain, strain background (Mus musculus, male and female)Ank1pale/pale
Ank1-KO		The Jackson LaboratoryJAX:009157See Materials and methods, Section Animals
Strain, strain background (Mus musculus, male and female)Ank1F/FThis paperJAX:036512See Materials and methods, Section Animals
Dr. Matthew Rasband (Baylor College of Medicine)
Strain, strain background (Mus musculus, male and female)Ank3F/FThe Jackson LaboratoryJAX:029797See Materials and methods, Section Animals
Sequence-based reagentGenotyping primer for Ank1F/F mouse (sense)This paperSee Materials and methods, Section Animals
5’-GGGAAACTCCACAGAGCCTGACGGGTCAGT-3’
Sequence-based reagentGenotyping primer for Ank1F/F mouse (missense)This paperSee Materials and methods, Section Animals
5’- GGC GTC CCTATGTTC CATCCTATA GATGACT-3’
Transfected construct (M. musculus)Full-length AnkR-GFPHo et al., 2014See Materials and methods, Section Plasmids
Transfected construct (M. musculus)β1 spectrin-MycHo et al., 2014See Materials and methods, Section Plasmids
Transfected construct (M. musculus)Full-length Kv3.1bDr. James Trimmer (University of California at Davis)See Materials and methods, Section Plasmids
Transfected construct (M. musculus)Full-length Kv3.1b-FlagThis paperSee Materials and methods, Section Plasmids p3XFLAG-CMV-7.1 vector
Transfected construct (M. musculus)Truncated Kv3.1b constructsThis paperSee Materials and methods, Section Plasmids p3XFLAG-CMV-7.1 vector
cell line (Homo-sapiens)HEK293TATCCSee Materials and methods, Section Immunoprecipitation
AntibodyAnkyrin-R
AnkR
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# 75–380RRID:AB_2491109IF (1:250)
IB (1:500)
IP (5 ul)
Antibodyβ1 spectrin
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# 73–374RRID:AB_2315814IF (1:250)
IB (1:1000)
IP (5 ul)
AntibodyAnkyrin-G
AnkG
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# 73–146RRID:AB_10697718IF (1:500)
AntibodyParvalbumin
Pv
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# 73–455RRID:AB_2629420IF (1:250)
AntibodyActin
(mouse monoclonal)		Millipore Cat# MAB1501RRID:AB_2223041IB (1:1000)
AntibodyTenascin-R
TnR
(mouse monoclonal)		R and D Systems Cat# MAB1624RRID:AB_2207001IF (1:500)
AntibodyAggrecan ACAN
Acan
(mouse monoclonal)		Millipore Cat# AB1031RRID:AB_90460IF (1:500)
AntibodyBrevican
BCAN
Bcan
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# 75–294RRID:AB_2315824IF (1:500)
AntibodyNrCAM
(mouse monoclonal)		R and D Systems Cat# MAB2034RRID:AB_2267411IF (1:500)
IB (1:1000)
IP (5 ul)
AntibodyKv3.1b
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# N16B/8RRID:AB_2750730IF (1:500)
IB (1:1000)
IP (5 ul)
AntibodyKv3.2
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# N410/17RRID:AB_2877619IF (1:250)
AntibodyKv3.3
(mouse monoclonal)		Antibodies-Online Cat# ABIN572016RRID:AB_10782137IF (1:500)
AntibodyKv7.2
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# N26A/23RRID:AB_2750761IF (1:500)
AntibodyPan sodium channel
PanNav
(mouse monoclonal)		UC Davis/NIH NeuroMab Facility Cat# N419/78RRID:AB_2877588IF (1:250)
AntibodyFlag-tag or DDDDK-tag
(mouse monoclonal)		MBL International Cat# M185-3LRRID:AB_11123930IB (1:1000)
AntibodyAnkyrin-R
AnkR
(rabbit polyclonal)		Ho et al., 2014RRID:AB_2833096IF (1:500)
IB (1:1000)
IP (5 ul)
AntibodyAnkyrin-R, AnkR, Ank1
(rabbit polyclonal)		Thermo Fisher Scientific Cat# PA5-63372RRID:AB_2638015IF (1:500)
IB (1:1000)
AntibodyNeurofilament M, NFM
(rabbit polyclonal)		Millipore Cat# AB1987RRID:AB_91201IB (1:2000)
AntibodySomatostatin, SST
(rabbit polyclonal)		Peninsula Laboratories Cat# T-4103.0050RRID:AB_518614IF (1:3000)
AntibodyParvalbumin Pv
(rabbit polyclonal)		Novus Cat# NB120-11427RRID:AB_791498IF (1:500)
AntibodyVersican,
VCAN
(rabbit polyclonal)		Millipore Cat# AB1032RRID:AB_11213831IF (1:500)
AntibodyPlexinA4, PlxnA4
(rabbit polyclonal)		Abcam Cat# ab39350RRID:AB_944890IF (1:500)
IB (1:1000)
IP (5 ul)
AntibodyNeruropilin-1, Nrp1
(rabbit polyclonal)		GeneTex Cat# GTX16786RRID:AB_422398IF (1:500)
IB (1:1000)
IP (5 ul)
AntibodyKv3.1 (rabbit polyclonal)LSBio (LifeSpan) Cat#LS-C322374-200RRID:AB_2891125IF (1:100)
AntibodyKv3.1b
(rabbit polyclonal)		Alomone Labs Cat# APC-014RRID:AB_2040166IF (1:500)
IB (1:1000)
IP (5 ul)
AntibodyKv3.3
(rabbit polyclonal)		Alomone Labs Cat# APC-102RRID:AB_2040170IF (1:500)
AntibodyGFP
(rabbit polyclonal)		Thermo Fisher Scientific, Cat# A-11122RRID:AB_221569IB (1:1000)
AntibodyNeurofascin, NF, NF186
(chicken polyclonal)		R and D Systems Cat# AF3235RRID:AB_10890736IF (1:500)
OtherFluorescein WFA, Wisteria floribunda agglutininVector Laboratories Cat# FL-1351RRID:AB_2336875Lectin
IF (1:250)
OtherFluorescein WFA, Wisteria floribunda agglutininBioworld Cat# 21761065–1RRID:AB_2833087Lectin
IF (1:100)
OtherTexas-Red WFA, Wisteria floribunda agglutininEY Laboratories Cat# F-3101–1RRID:AB_2315605Lectin
IF (1:250)
Software, algorithmFiji, Image JNIHRRID:SCR_002285
Software, algorithmPrismGraph PadRRID:SCR_011323

  1. IF: Immunofluorescence; IB: Immunoblot; IP:Immunoprecipitation

Additional files

Supplementary file 1

Intrinsic properties of WFA+ cells in Ank1F/F, Ank1+/+;Dlx5/6-Cre, and Ank1F/F;Dlx5/6-Cre mice.

Data are from 3 Ank1F/F, 2 Ank1+/+;Dlx5/6-Cre, and 4 Ank1F/F;Dlx5/6-Cre mice, and are reported as mean ± SEM (number of cells). Bolded p values indicate significance.

https://cdn.elifesciences.org/articles/66491/elife-66491-supp1-v2.docx
Supplementary file 2

Abbreviations used throughout the paper.

https://cdn.elifesciences.org/articles/66491/elife-66491-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/66491/elife-66491-transrepform-v2.docx

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