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Disordered breathing in a mouse model of Dravet syndrome

  1. Fu-Shan Kuo
  2. Colin M Cleary
  3. Joseph J LoTurco
  4. Xinnian Chen
  5. Daniel K Mulkey  Is a corresponding author
  1. University of Connecticut, United States
Research Article
Cite this article as: eLife 2019;8:e43387 doi: 10.7554/eLife.43387
6 figures, 4 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Conditional expression of Scn1a A1783V in inhibitory neurons results in premature death.

(A) Construct design and validation of A1783V variant transcript expression. Note that this line was developed by Ana Mingorance (Chief Development Officer of the Loulou Foundation) and is available at JAX (sock # 026133). Ai, schematic shows loxP sites flanking wild type exon 26 followed by an edited version of exon 26 that contains the human A1783V pathological variant (ΔE26). When Cre recombinase is expressed, wild type exon 26 is removed, thus allowing transcription of ΔE26. Aii, Agarose gel shows detectable levels of Scn1a transcript (expected size of 831 bp) in brainstem tissue isolated from each genotype (primers span between exon 25 and 26, including residue 1783 of exon 26). Water was used as a no template negative control. Aiii, PCR products were sequenced to confirm that transcript containing A1783V is detectable in 40% of samples from Scn1aΔE26 tissue but was not detectable in samples from Slc32a1cre/+ and Scn1afl/+ control tissue. (B–C), fluorescent in situ hybridization (RNAScope) was performed to characterize expression of Scn1a transcript in inhibitory (Slc32a1+, Slc32a1) and glutamatergic (Vglut2+, Slc17a6) neurons in the RTN region in brainstem sections from control and Scn1aΔE26mice. (B) brainstem sections from Slc32a1cre/+ and Scn1aΔE26 mice containing the RTN show Scn1a labeling (green puncta) of both Vgat+ and Vglut2 +neurons. (C), summary data show Scn1a transcript expression (normalized to cell size) in Vgat+ and Vglut2 +RTN neurons from each genotype; channel transcript was reduced in Vgat+ cells from Scn1aΔE26 mice (0.43 ± 0.7 mRNA/area, n = 94 cells) compared to control (0.73 ± 0.9 mRNA/area, n = 82 cells) (p<0.05), whereas Vglut2 +cells showed low channel transcript across both genotypes. (D–E) Scn1aΔE26 mice did not show any obvious differences gross morphology (A) body weight (D) or temperature (E) compared to age-matched litter mate control mice. (F) (Figure 1—source data 1), survival curve shows that control mice (n = 57) survive to adulthood (30 days postnatal) while Scn1aΔE26 mice (n = 41) die prematurely starting at 9 days postnatal and reaching 100% lethality by 25 days (χ2 = 63.9, p<0.0001). These results were compared using a two-way ANOVA and Sidak multiple comparison test. *, p<0.05; ***p<0.001; ****p<0.0001.

https://doi.org/10.7554/eLife.43387.002
Figure 1—source data 1

Survival curves for Slc32a1cre/+ and Scn1aΔE26 mice.

https://doi.org/10.7554/eLife.43387.004
Figure 1—figure supplement 1
Breeding strategy to generate mice that heterologously express the Scn1a A1783V pathological variant conditionally in inhibitory neurons (Scn1aΔE26 mice).

Homozygous Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J reporter mice (Ai14; JAX no. 007914) on a 100% C57BL/6J background are bred with mice that express Cre recombinase targeted to Slc32a1, the gene encoding Vgat (Slc32a1Cre mice, JAX no. 016962), on a mixed background of 75%::25% C57BL/6J::129/SvJ to produce Slc32a1Cre:: TdT double-heterozygous mice with a 85% C57BL6/J: : 15% 129/SvJ background. These mice are then bred with Scn1aA1783Vfl/+ (JAX no. 026133) maintained on a 100% C57BL/6J background to produce experimental animals with a genotype of Slc32a1cre+/-::TdT+/-::Scn1afl/+ (Scn1aΔE26) and control animals of the following genotypes Slc32a1cre-/-::TdT+/-::Scn1afl/+ (Scn1afl/+) and Slc32a1cre+/-::TdT+/-::Scn1a+/+ (Slc32a1cre/+). Experimental and control mice had a common background of 90% C57BL6/J: : 10% 129/SvJ. The proportion of each background stain was determined by Genome scan analysis (JAX).

https://doi.org/10.7554/eLife.43387.003
Scn1aΔE26 exhibit frequent spontaneous seizures.

(A) traces of raw EcoG activity show that Scn1aΔE26 mice but not Slc32a1cre/+ mice exhibit frequent spontaneous burst of high amplitude poly-spike activity. The arrow identifies a typical seizure-like poly-spike event that was analyzed further by power spectral analysis in panel D. Polyspike events with a minimum duration of 14 ms were accompanied by seizure-like behavior and so were considered epileptic activity. (B), Scn1aΔE26 mice showed more frequent epileptic poly-spike bursts of activity (Bi, dotted line designates duration threshold for epileptic activity); poly-spike bursts (>14 ms) occurred more frequently in Scn1aΔE26 mice (Bi-Bii) (control 0.13 ± 0.1 events/2 hr, n = 6; Scn1aΔE26 0.37 ± 0.05 events/2 hr, n = 6; T10 = 3.009, p<0.01) and lasted for a longer duration (Biii) (control 7.6 ± 0.6 ms, n = 6; Scn1aΔE26 15.6 ± 0.8 ms, n = 6, T10 = 2.268, p<0.05) compared to control animals. Ci, representative power spectrum density (PSD) plots of spontaneous poly-spike burst events show typical strong activity in the theta-, alpha and beta frequency range in Scn1aΔE26 but not control mice. Cii-Ciii (Figure 2—source data 1), summary data (normalized to the maximum value at each event) show PSD peak (Cii) and PSD area under the curve (Ciii) of each frequency range for each genotype. Note that poly-spike burst events measured in Scn1aΔE26 mice show increased activity in the theta, alpha and beta range. Di-iii, poly-spike burst events recorded from a Scn1aΔE26 mouse (arrow in panel A) plotted on an expanded time scale (Di) and corresponding time frequency distribution (Dii) and deconstructed spectrum into its various frequency domains (Diii). These results were compared using a two-way ANOVA and the Sidak multiple comparison test. *, p<0.05; **, p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.43387.008
Figure 2—source data 1

Plots of PSD peak and PSD area of poly-spike burst events measured in Slc32a1cre/+ and Scn1aΔE26 mice.

https://doi.org/10.7554/eLife.43387.009
Scn1aΔE26 mice show reduced respiratory output under control conditions and during exposure to high CO2.

For these experiments Scn1afl/+ and Slc32a1cre/+ were used as control. (A) traces of respiratory activity from a control and Scn1aΔE26 mouse during exposure to room air, 100% O2 and 3–7% CO2 (balance O2). (B–D), summary data (n = 22 control; n = 17 Scn1aΔE26) show respiratory frequency (B), tidal volume (C) and minute ventilation (D) are reduced in Scn1aΔE26 mice compared to control under room air conditions. (E), traces of respiratory activity (left) and summary data (right) show that under room air conditions both control and Scn1aΔE26 mice exhibit periods of apnea; the frequency of these events were similar between genotypes, however, they lasted for a longer duration in Scn1aΔE26 mice compared to control. F-H (Figure 3—source data 1), summary data shows the respiratory frequency (F), tidal volume (G) and minute ventilation response of control and Scn1aΔE26 mice to graded increases in CO2 (balance O2). Scn1aΔE26 mice showed a blunted respiratory frequency to 5% and 7% CO2 which resulted in a diminished CO2/H+-dependent increase in minute ventilation. These results were compared using either unpaired t test (panels B-E) or two-way ANOVA followed by the Holm-Sidak multiple comparison test (panels F-H). *, difference between means p<0.05, #, different interaction factor, p<0.05.

https://doi.org/10.7554/eLife.43387.010
Figure 3—source data 1

Summary data showing respiratory frequency, tidal volume and minute ventilatory responses to CO2 in control and Scn1aΔE26 mice.

https://doi.org/10.7554/eLife.43387.011
Brainstem inhibitory neurons in slices from Scn1aΔE26 show diminished basal activity and repetitive firing behavior during sustained depolarization.

(A) segments of membrane potential from inhibitory neurons in the RTN region in slices from control and Scn1aΔE26 mice during depolarizing current injections (40 to 220 pA; 1 s duration) from a membrane potential of –80 mV. (B) summary data shows inhibitory neurons in slices from Scn1aΔE26 mice (n = 36) are less active under resting conditions (0 pA holding current) compared to inhibitory neurons in slices form Slc32a1cre/+ control mice (n = 26 cells). (C), summary data and representative voltage responses to a −60 pA current injection show that inhibitory neurons from each genotype had similar input resistance. (D) (Figure 4—source data 1), input-output relationship show that inhibitory neurons from Scn1aΔE26 mice generate fewer action potentials in response to moderate depolarizing current injections (1 s duration) and at more positive steps go into depolarizing block. Results were compared using t-test (B–C) and two-way ANOVA and Sidak multiple comparison test (D). *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.43387.012
Figure 4—source data 1

Evoked firing responses of inhibitory neurons in slices from Slc32a1cre/+ and Scn1aΔE26 mice.

https://doi.org/10.7554/eLife.43387.013
The Scn1a A1783V pathological variant may result in loss of channel function by increased voltage-dependent inactivation.

(A) average spontaneous action (Slc32a1cre/+ control n = 24 spikes, Scn1aΔE26 n = 29 spikes (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and Scn1aΔE26 mice (Ai) and corresponding phase plot (Aii) (dV/dt; Y-axis vs mV; X-axis) of the traces in panel Ai show that cells expressing Scn1a A1783V depolarize slower compared to control cells (Figure 5—source data 1). (B), average first action potential following a hyperpolarizing pre-potential (−100 pA; 1 s) (Slc32a1cre/+ control n = 13 spikes, Scn1aΔE26 n = 16 spikes) (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and Scn1aΔE26 mice (Bi) and corresponding phase plot (Bii) of traces in panel Bi show that holding cells at a negative pre-potential to remove sodium channel inactivation improved the depolarization kinetics of subsequent spikes (Figure 5—source data 1). (C), average first action potential following a depolarizing pre-potential (+180 pA; 1 s) (control n = 9 spikes, Scn1aΔE26 n = 11 spikes (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and Scn1aΔE26 mice (Ci) and corresponding phase plot (Cii) of traces in panel Ci show that holding cells at a depolarized pre-potential to increase sodium channel inactivation diminished genotype differences in action potential kinetics (Figure 5—source data 1). (D–F), summary data showing the maximum rate of depolarization (D), action potential amplitude (E) and action potential threshold (F) of spontaneous action potentials and first spikes following positive or negative pre-potentials recorded in slices from control and Scn1aΔE26 mice. Results were compared by two-way ANOVA and the Sidak multiple comparison test.. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

https://doi.org/10.7554/eLife.43387.014
Figure 5—source data 1

Phase plots of spontaneous action potentials and the first spike elicited following positive or negative pre-potentials in slices from Slc32a1cre/+ and Scn1aΔE26 mice.

https://doi.org/10.7554/eLife.43387.015
Chemosensitive RTN neurons in slices from Scn1aΔE26 mice are hyper-excitable.

(A) firing rate traces from chemosensitive neurons in slices from control (top) and Scn1aΔE26 mice (bottom) show that neurons from both genotypes respond to changes in CO2/H+; RTN neurons are spontaneously active under control conditions (5% CO2; pHo 7.3) and respond to 7% CO2 (pHo 7.2) and 10% CO2 (pHo 7.0) with a linear increase in activity, whereas exposure to 3% CO2 (pHo 7.6) decreases neural activity. However, basal activity and CO2/H+-dependent output of RTN chemoreceptors from Scn1aΔE26 tissue is enhanced compared to RTN neurons in slices from Slc32a1cre/+control mice. (B) double-immunolabeling shows that a Lucifer Yellow-filled CO2/H+-sensitive RTN neuron (green) is immunoreactive for phox2b (magenta), the merged image is shown to the right. We confirmed that all CO2/H+-sensitive neurons (Slc32a1cre/+ control n = 12; Scn1aΔE26 n = 11) included in this study were phox2b-positive. (C–D) (Figure 6—source data 1), summary data shows that RTN chemoreceptors in slices from Scn1aΔE26 mice have higher basal activity (C) and enhanced CO2/H+-dependent output between 3–10% CO2 (D). Results were compared by t-test (C) or ANCOVA test (D). *, p<0.05.

https://doi.org/10.7554/eLife.43387.016
Figure 6—source data 1

CO2/H+-evoked activity in chemosensitive RTN neurons in slices from Slc32a1cre/+ and Scn1aΔE26 mice.

https://doi.org/10.7554/eLife.43387.017

Tables

Table 1
Behavioral Assessment of Seizure activity.
https://doi.org/10.7554/eLife.43387.005
Racine scoreN012345Behavioral arrest
Slc32a1cre/+2217500000
Scn1aΔE26220336235
Table 2
Febrile seizure propensity.
https://doi.org/10.7554/eLife.43387.006
GenotypeNWeightInduced seizure (%)
Slc32a1cre/+106.65 ± 0.30
Scn1aΔE2696.71 ± 0.3100****
  1. ****Fisher’s exact test p<0.000

Table 2—source data 1

Febrile seizure propensity.

https://doi.org/10.7554/eLife.43387.007
Key resources table
Reagent typeDesignationSource or referenceIdentifiersAdtl. info
Strain, strain
background (M. musculus, Scn1a A1783V, C57BL6/J background)
B6(Cg)-Scn1atm1.1Dsf/JJackson LaboratoriesRRID:IMSR_JAX:026133unpublished model
Strain, strain background (M. musculus, Vgat-iris-Cre, mixed 129/SvJ
and C57BL6/J background)
Slc32a1tm2(cre)Lowl/JPMID: 21745644RRID:IMSR_JAX:016962
Strain, strain background
(M. musculus, tdTomato reporter Ai14, C57BL6/J background)
B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JPMID: 20023653RRID:IMSR_JAX:007914
Genetic reagent (M-MLV Reverse Transcriptase (200 U/µL))MMLV RT first-strand reagentThermoFisher Scientific28025013
Genetic reagent (GoTaq Flexi DNA Polymerase)Taq polymerasePromegaM8291
Antibodygoat anti-Phox2b antibodyR and D SystemsAF4940; RRID:AB_10889846used on fixed tissue, 1:500 dilution
Antibodyrabbit anti-Lucifer Yellow antibodyInvitrogenA-5750; RRID:AB_2536190used on fixed tissue, 1:2000 dilution
Sequence based reagentRNAscope Probe- Mm-Scn1aACDBio4341811:50
Sequence based reagentRNAscope Probe- Mm-Slc32a1-C2ACDBio319191-C21:50
Sequence based reagentRNAscope Probe- Mm-Slc17a6-C2ACDBio319171-C21:50
Commercial assay or kitRNAscope Fresh Frozen Multiplex Fluorescent KitACDBio320851
Commercial
assay or kit
NEB PCR Cloning KitNew England BioLabsE1202S
Commercial assay or kitQIAprep Gel Extraction KitQiagen28704
Commercial assay or kitQIAprep Spin Miniprep KitQiagen27104
Commercial
assay or kit
Direct-zol RNA MicroPrepZymo ResearchR2061
Chemical compound, drugLucifer YellowSigmaB42610.10%
Software, algorithmSnapGene ViewerSnapGeneRRID:SCR_015053
Software, algorithmPonemahDSIRRID:SCR_017107Version 5.20
Software, algorithmSpikeCambridge
Electronic Design
RRID:SCR_000903Version 5.0
Software, algorithmSireniaPinnacile TechnologyRRID:SCR_016183
Software, algorithmMatlabMathworksRRID:SCR_001622Version R2018
Software, algorithmBrainstormTadel et al., 2011RRID:SCR_001761Version 3.0
Software, algorithmPrism 7GraphPadRRID:SCR_002798Version 7.03
Software, algorithmpCLAMP 10Molecular DevicesRRID:SCR_011323Version 10
Software, algorithmImageJNIHRRID:SCR_003070Version 2.0.0
Table 3
Probes used for FISH.
https://doi.org/10.7554/eLife.43387.018
Gene nameProbe cat no.Target region
Scn1a4341811624–2967
Slc32a1319191-C2894–2037
Slc17a6319171-C21986–2998

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