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Deletion of KCNQ2/3 potassium channels from PV+ interneurons leads to homeostatic potentiation of excitatory transmission

  1. Heun Soh
  2. Suhyeorn Park
  3. Kali Ryan
  4. Kristen Springer
  5. Atul Maheshwari  Is a corresponding author
  6. Anastasios V Tzingounis  Is a corresponding author
  1. University of Connecticut, United States
  2. Baylor College of Medicine, United States
Short Report
Cite this article as: eLife 2018;7:e38617 doi: 10.7554/eLife.38617
4 figures, 1 table and 1 additional file

Figures

Ablation of Kcnq2/3 channels from PV+/SST+interneurons leads to increased excitatory transmission.

(a) Top, representative sEPSC and sIPSC traces recorded from mouse CA1 pyramidal neurons (P15–P19) in acute hippocampal slices from control (number of animals = 8) and either Kcnq2/3 interneuron (IN:Kcnq2/3 null; number of animals = 4) or Kcnq2 pyramidal neuron conditional knockout (PYR:Kcnq2 null; number of animals = 3) mice. Bottom, cumulative distribution plots of sIPSC and sEPSC inter-event intervals recorded in pyramidal neurons of IN:Kcnq2/3 null and PYR:Kcnq2 null mice. Insets: summary graphs of average inter-event frequency. Statistical comparisons were performed with a one-way analysis of variance (ANOVA; p<0.05) followed by a Fisher LSD post-hoc test (*: p<0.05, **: p<0.001). For comparing sEPSC frequency: ANOVA F(2,29) =5.168, p=0.012; for control vs. PYR:Kcnq2 null p=0.0051; for control vs. IN:Kcnq2/3 null p=0.038. (b) Top, representative mEPSC and mIPSC traces recorded from mouse CA1 pyramidal neurons from control (number of animals = 6), IN:Kcnq2/3 null (number of animals = 3) or PYR:Kcnq2 null (number of animals = 4) mice, respectively. Bottom, cumulative distribution plots of mIPSCs and mEPSCs inter-event intervals recorded in pyramidal neurons of IN:Kcnq2/3 null and PYR:Kcnq2 null mice. Insets: summary graphs of average inter-event frequency. Statistical comparisons were performed with one-way ANOVA followed by Fisher LSD post-hoc test (*: p<0.05, **: p<0.001). For comparing sEPSC frequency ANOVA F(2,22) =10.74, p=0.0006; for control vs. PYR:Kcnq2 null p=0.3764; for control vs. IN:Kcnq2/3 null p=0.0002. Each data point represents recording from one pyramidal neuron. Data in summary graphs are represented as mean and s.e.m.

https://doi.org/10.7554/eLife.38617.002
Figure 2 with 3 supplements
Loss of KCNQ2/3 activity leads to increased excitability of PV+interneurons.

(a) Top, representative voltage responses from a +150 pA current injection step (1 s) in PV- and SST-like interneurons in either the CA1 region of the hippocampus (P12–P17) or L2/3 of the somatosensory cortex (P8–P11). For L2/3 recordings, cells were also confirmed by immunoreactivity against SST antibodies. Bottom, summary graphs showing the effect of deleting KCNQ2 and KCNQ3 channels on action potential number from CA1 PV-like (control n = 8/6; IN:Kcnq2/3 null n = 8/5), SST-like (control n = 19/8; IN:Kcnq2/3 null n = 8/4), and L2/3 (PV-like: control n = 10/7; IN:Kcnq2/3 null n = 8/4; SST-like: control n = 10/6; IN:Kcnq2/3 null n = 5/4) interneurons (Vh=-75 to −77 mV). For CA1 PV-like cells (P16–P25), F(9,126)=2.849, p=0.0043; for L2/3 PV-like cells, F(9,144)=3.845, p=0.0002); for CA1 SST-like cells (P15–P19), F(9,225)=0.601, p=0.7955; and for L2/3 SST-like cells, F(9,117)=0.326, p=0.965. Significance was determined using a two-factor mixed ANOVA. See Figure 2—figure supplement 1 showing that indeed SST cells express KCNQ2 and KCNQ3 mRNA. (b) Top, representative voltage responses to a series of current injection steps (1 s) in PV+ and SST+ interneurons in the CA1 region of the hippocampus (Vh=-75 to −77 mV). Bottom left, summary graph showing the effect of deleting KCNQ2 and KCNQ3 channels on action potential number from CA1 PV+ cells (control n = 15/8; PV:Kcnq2/3 null n = 14/7; F(9,243)=3.558 with p=0.0004). Middle left, summary graph showing that loss of KCNQ2/3 channels decreases PV+ input resistance (control, n = 15/8; PV:Kcnq2/3 null, n = 14/7; df = 27 t=−2.54 p=0.017 unpaired Student’s t-test). See also Figure 2—figure supplement 2 regarding PV+ Kcnq2/3 null neurons diversity of intrinsic properties. Middle right, summary graph showing the effect of deleting KCNQ2 and KCNQ3 channels on action potential number from CA1 SST+ cells (control n = 6/2; SST:Kcnq2/3 null n = 8/4; F(9,108)=0.729 with p=0.6814). Bottom right, summary graph showing loss of KCNQ2/3 channels did not decrease SST+ input resistance (control n = 6/2; SST:Kcnq2/3 null, n = 8/4; df = 12 t=−0.42 p=0.68 unpaired Student’s t-test). ‘n’ designates number of cells followed by number of animals. Each data point represents recording from one neuron. Data in summary graphs are represented as mean and s.e.m.

https://doi.org/10.7554/eLife.38617.004
Figure 2—figure supplement 1
FISH shows presence of KCNQ2 and KCNQ3 in SST+interneurons.

Confocal micrographs of coronal sections of CA1 region of wild-type mice. Micrographs shows co-localization of mRNA for somatostatin, KCNQ2 and KCNQ3. All micrographs have been counterstained with DAPi (blue).

https://doi.org/10.7554/eLife.38617.005
Figure 2—figure supplement 2
PV:Kcnq2/3 null interneurons could differ in their intrinsic excitability properties.

Top, representative voltage responses to a series of current injection steps (1 s) in PV+ interneurons in the CA1 region of the hippocampus (P16–P25). We found that Kcnq2/3 null PV+ interneurons could exhibit two intrinsic excitability behaviors. Type I that has a sag ratio of 0.9 and greater, similar to control neurons, and type II that has a sag ratio of 0.8 or less. Excluding type II cells (red; as in Figure 2b) or including all types of cells (blue) as shown here, it did not change the conclusions of this work. The lower sag ratio might indicate immature PV+ cells, however, other possibilities cannot be excluded at this point.

https://doi.org/10.7554/eLife.38617.006
Figure 2—figure supplement 3
The pan-KCNQ blocker XE991 increases PV+interneuron excitability.

Left, representative voltage responses from a + 200 pA current injection step (1 s; Vh=-75 to −77 mV) in PV+ interneurons (Pvalb-Cre;Kcnq2+/+;Ai9) from the CA1 region of the hippocampus (P23–P25) before and after application of 20 μM XE-991 (15 min application). Right, summary graphs showing the of XE-991 on action potential number (n = 6/3; F(1,5)=38.379, p=0.0016) and input resistance (control 169 ± 20 MΩ,+XE-991 143 ± 20 MΩ, n = 6/3; t = 1.355 df = 5 p=0.2335). Significance was determined using two-way factor repeated measures ANOVA and two-tailed paired Student’s t-test for action potential number and input resistance, respectively.

https://doi.org/10.7554/eLife.38617.008
Ablation of Kcnq2 from PVinterneurons leads to increased excitatory transmission in pyramidal neurons.

For simplicity we refer Pvalb-Cre;Kcnq2;Ai9 mice in the figure as Pvalb-Cre;Kcnq2f/f or Pvalb-Cre;Kcnq2+/+. (a) Left, representative voltage responses from a + 200 pA current injection step (1 s; Vh= −75 to −77 mV) in PV+ interneurons from the CA1 region of the hippocampus (P23–P25). Right, summary graph showing the effect of deleting Kcnq2 on action potential number from PV+ interneurons (Pvalb-Cre;Kcnq2+/+;Ai9 n = 15/4; Pvalb-Cre;Kcnq2f/f;Ai9 n = 14/3; F(9,243)=3.558, p=0.0004). Significance was determined using a two-factor mixed ANOVA. (b) Left, representative voltage responses from a + 200 pA current injection step (1 s; Vh=-75mV) in pyramidal neurons from the CA1 region of the hippocampus (P30–P32). Right, summary graph showing the effect of deleting Kcnq2 from pyramidal neurons in action potential number (Pvalb-Cre;Kcnq2+/+;Ai9 n = 15/3; Pvalb-Cre;Kcnq2f/f;Ai9 n = 12/2; F(9,225)=0.4891, p=0.88). Significance was determined using a two-factor mixed ANOVA. (c) Left, representative sEPSC traces recorded from CA1 pyramidal neurons (P32–P35) in acute hippocampal slices from control and Kcnq2 null PV+ interneurons. Right, summary bar graphs of sEPSC frequency (Pvalb-Cre;Kcnq2+/+;Ai9 1.165 ± 0.12 Hz, n = 13/4; Pvalb-Cre;Kcnq2f/f;Ai9: 2.108 ± 0.22 Hz, n = 19/3; p=0.0098 Mann-Whitney test) and amplitude (Pvalb-Cre;Kcnq2+/+;Ai9 15.9 ± 0.86 pA, n = 13/4; Pvalb-Cre;Kcnq2f/f;Ai9: 15.3 ± 0.68 pA, n = 19/3; DF = 30 t = 0.62 p=0.5394). Statistical comparisons were performed with two-tailed unpaired Student’s t-test or Mann-Whitney when the variance between the two groups was significantly different. ‘n’ designates number of cells followed by number of animals. Each data point represents recording from one neuron. Data in summary graphs are represented as mean and s.e.m.

https://doi.org/10.7554/eLife.38617.011
In vivo hyperexcitability with loss of Kcnq2 in PV-expressing interneurons.

(a) Using simultaneous video-EEG monitoring, Stage five onset was defined as the latency to rearing and falling with forelimb clonus, associated with bilateral epileptiform activity on EEG. (b) Loss of one or both Kcnq2 alleles in PV-expressing interneurons led to significantly reduced latency to seizure-onset (p=0.0370 and p=0.0047, respectively, Log-rank (Mantel-Cox) test; Pvalb-cre;Kcnq2+/+ n = 14; Pvalb-cre;Kcnq2f/+ n = 13; Pvalb-cre;Kcnq2f/f n = 12). ‘n’ designates number of animals. Bar = 1 min, inset, 5 s.

https://doi.org/10.7554/eLife.38617.013

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Kcnq2NANCBI_Gene:16536;
MGI:1309503
Gene (Mus musculus)Kcnq3NANCBI_Gene:110862;
MGI:1336181
Strain, strain
background (M. musculus,
Emx1IRESCre, C57BL/6J
background)
B6.129S2-Emx1tm1(cre)Krj /JPMID: 12151506RRID:IMSR_JAX:005628
Strain, strain
background (M. musculus,
Nkx2.1-Cre,
C57BL/6J background)
C57BL/6J-Tg
(Nkx2-1-cre)2Sand/J
PMID: 17990269RRID:IMSR_JAX:008661
Strain, strain
background (M. musculus,
Sst-IRES-Cre, C57BL/6J
background)
Ssttm2.1(cre)Zjh/JPMID: 21943598RRID:IMSR_JAX:013044
Strain, strain
background (M. musculus,
Pvalb-Cre, C57BL/6J
background)
B6;129P2-Pvalbtm1(cre)Arbr/JPMID: 15836427RRID:IMSR_JAX:008069
Strain, strain
background (M. musculus,
tdTomato reporter Ai9,
C57BL/6J background)
B6.Cg-Gt(ROSA)
26Sortm9(CAG-tdTomato)Hze/J
PMID: 20023653RRID:IMSR_JAX:007909
Strain, strain
background (M. musculus,
Kcnq2f/f, C57BL/6J
background)
Kcnq2f/fPMID: 24719109N-A
Strain, strain
background (M. musculus,
Kcnq3f/f, C57BL/6J
background)
Kcnq3f/fPMID: 24719109N-A
AntibodyAlexa fluor 488
streptavidin
InvitrogenInvitrogen:S32354;
RRID:AB_2315383
(1:500)
Antibodyanti-Lucifer yellow
(rabbit polyclonal)
InvitrogenInvitrogen:A5750;
RRID:AB_2536190
(1:500)
Antibodyanti-Somatostatin
(rat monoclonal)
MilliporeMillipore:MAB354;
RRID:AB_2255365
(1:250)
AntibodyAlexa fluor 488
anti-rabbit secondary
(goat polyclonal)
InvitrogenInvitrogen:A11034;
RRID:AB_2576217
(1:500)
AntibodyAlexa fluor 647
anti-rat secondary
(goat polyclonal)
InvitrogenInvitrogen:A21247;
RRID:AB_141778
(1:500)
Sequence-based
reagent
somatostatin mRNA
probe (mouse); Mm-Sst-C1
ACDBioCat#:404631(1:50)
Sequence-based
reagent
parvalbumin mRNA
probe (mouse);
Mm-Pvalb-C1
ACDBioCat#:421931(1:50)
Sequence-based
reagent
tdTomato mRNA probe
(mouse); Mm-tdTomato-C3
ACDBioCat#:317041-C3(1:50)
Sequence-based
reagent
Kcnq2 mRNA probe
(mouse); Mm-Kcnq2-O1
ACDBio; this paperCat#:300031-C2(1:50); custom made
probe that targets exons
2–5 of Kcnq2
Sequence-based
reagent
Kcnq3 mRNA probe
(mouse); Mm-Kcnq3-O1
ACDBio; this paperCat#:300031-C3(1:50); custom made
probe that targets exons
2–5 of Kcnq2
Commercial
assay or kit
RNAscope Fresh Frozen
Multiplex Fluorescent kit
ACDBioCat#:320851
Chemical
compound, drug
CNQXAbcamab120017
Chemical
compound, drug
D-AP5Abcamab120003
Chemical
compound, drug
PicrotoxinAbcamab120315
Chemical
compound, drug
Tetrodotoxin; TTXAbcamab120054
Chemical
compound, drug
XE-991Abcamab120089
Chemical
compound, drug
Lucifer YellowSigmaCat#:B4261(0.1%)
Chemical
compound, drug
BiocytinMolecular ProbesCat#:L1177(0.05%)
Software, algorithmPrism 7GraphPadRRID:SCR_002798Version 7.03
Software, algorithmClampfit 10Molecular DevicesRRID:SCR_011323
Software, algorithmMinianalysisSynaptosoftRRID:SCR_002184
Software, algorithmOrigin 8 ProOriginLabRRID:SCR_014212Version 8.0951
Software, algorithmImageJNIHRRID:SCR_003070Version 2.0.0

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