Figures and data in Disordered breathing in a mouse model of Dravet syndrome

Version of Record: May 8, 2019
Accepted Manuscript: April 26, 2019

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Fu-Shan Kuo

Colin M Cleary

Joseph J LoTurco

Xinnian Chen

Daniel K Mulkey

(2019)
Disordered breathing in a mouse model of Dravet syndrome

eLife 8:e43387.

https://doi.org/10.7554/eLife.43387
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Figures
Tables
Additional files

6 figures, 4 tables and 1 additional file

Figures

Figure 1 with 1 supplement

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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 …
https://doi.org/10.7554/eLife.43387.002

Figure 1—source data 1 Survival curves for Slc32a1^cre/+ and Scn1a^ΔE26 mice.: https://doi.org/10.7554/eLife.43387.004
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Figure 1—figure supplement 1

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Breeding strategy to generate mice that heterologously express the *Scn1a* A1783V pathological variant conditionally in inhibitory neurons (Scn1a^ΔE26 mice).

Homozygous Gt(ROSA)26Sor^{tm14(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 …
https://doi.org/10.7554/eLife.43387.003

Figure 2

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*Scn1a^ΔE26* exhibit frequent spontaneous seizures.

(A) traces of raw EcoG activity show that *Scn1a^ΔE26* mice but not *Slc32a1*^cre/+ mice exhibit frequent spontaneous burst of high amplitude poly-spike activity. The arrow identifies a typical …
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 Slc32a1^cre/+ and Scn1a^ΔE26 mice.: https://doi.org/10.7554/eLife.43387.009
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Figure 3

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*Scn1a^ΔE26* mice show reduced respiratory output under control conditions and during exposure to high CO₂.

For these experiments *Scn1a^fl/+* and *Slc32a1*^cre/+ were used as control. (A) traces of respiratory activity from a control and *Scn1a^ΔE26* mouse during exposure to room air, 100% O₂ and 3–7% CO₂ …
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 Scn1a^ΔE26 mice.: https://doi.org/10.7554/eLife.43387.011
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Figure 4

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Brainstem inhibitory neurons in slices from *Scn1a^ΔE26* 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 *Scn1a^ΔE26* mice during depolarizing current injections (40 to 220 pA; 1 s duration) from a …
https://doi.org/10.7554/eLife.43387.012

Figure 4—source data 1 Evoked firing responses of inhibitory neurons in slices from Slc32a1^cre/+ and Scn1a^ΔE26 mice.: https://doi.org/10.7554/eLife.43387.013
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Figure 5

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The *Scn1a* A1783V pathological variant may result in loss of channel function by increased voltage-dependent inactivation.

(A) average spontaneous action (*Slc32a1*^cre/+ control n = 24 spikes, *Scn1a^ΔE26* n = 29 spikes (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in …
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 Slc32a1^cre/+ and Scn1a^ΔE26 mice.: https://doi.org/10.7554/eLife.43387.015
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Figure 6

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Chemosensitive RTN neurons in slices from *Scn1a^ΔE26* mice are hyper-excitable.

(A) firing rate traces from chemosensitive neurons in slices from control (top) and *Scn1a^ΔE26* mice (bottom) show that neurons from both genotypes respond to changes in CO₂/H⁺; RTN neurons are …
https://doi.org/10.7554/eLife.43387.016

Figure 6—source data 1 CO₂/H⁺-evoked activity in chemosensitive RTN neurons in slices from Slc32a1^cre/+ and Scn1a^ΔE26 mice.: https://doi.org/10.7554/eLife.43387.017
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Tables

Table 1

Behavioral Assessment of Seizure activity.

https://doi.org/10.7554/eLife.43387.005

Racine score	N	0	1	2	3	4	5	Behavioral arrest
Slc32a1^cre/+	22	17	5	0	0	0	0	0
Scn1a^ΔE26	22	0	3	3	6	2	3	5

Table 2

Febrile seizure propensity.

https://doi.org/10.7554/eLife.43387.006

Genotype	N	Weight	Induced seizure (%)
Slc32a1^cre/+	10	6.65 ± 0.3	0
Scn1a^ΔE26	9	6.71 ± 0.3	100****

^****Fisher’s exact test p<0.000

Table 2—source data 1 Febrile seizure propensity.: https://doi.org/10.7554/eLife.43387.007
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Key resources table

Reagent type	Designation	Source or reference	Identifiers	Adtl. info
Strain, strain background (M. musculus, Scn1a A1783V, C57BL6/J background)	B6(Cg)-Scn1atm1.1Dsf/J	Jackson Laboratories	RRID:IMSR_JAX:026133	unpublished model
Strain, strain background (M. musculus, Vgat-iris-Cre, mixed 129/SvJ and C57BL6/J background)	Slc32a1tm2(cre)Lowl/J	PMID: 21745644	RRID:IMSR_JAX:016962
Strain, strain background (M. musculus, tdTomato reporter Ai14, C57BL6/J background)	B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	PMID: 20023653	RRID:IMSR_JAX:007914
Genetic reagent (M-MLV Reverse Transcriptase (200 U/µL))	MMLV RT first-strand reagent	ThermoFisher Scientific	28025013
Genetic reagent (GoTaq Flexi DNA Polymerase)	Taq polymerase	Promega	M8291
Antibody	goat anti-Phox2b antibody	R and D Systems	AF4940; RRID:AB_10889846	used on fixed tissue, 1:500 dilution
Antibody	rabbit anti-Lucifer Yellow antibody	Invitrogen	A-5750; RRID:AB_2536190	used on fixed tissue, 1:2000 dilution
Sequence based reagent	RNAscope Probe- Mm-Scn1a	ACDBio	434181	1:50
Sequence based reagent	RNAscope Probe- Mm-Slc32a1-C2	ACDBio	319191-C2	1:50
Sequence based reagent	RNAscope Probe- Mm-Slc17a6-C2	ACDBio	319171-C2	1:50
Commercial assay or kit	RNAscope Fresh Frozen Multiplex Fluorescent Kit	ACDBio	320851
Commercial assay or kit	NEB PCR Cloning Kit	New England BioLabs	E1202S
Commercial assay or kit	QIAprep Gel Extraction Kit	Qiagen	28704
Commercial assay or kit	QIAprep Spin Miniprep Kit	Qiagen	27104
Commercial assay or kit	Direct-zol RNA MicroPrep	Zymo Research	R2061
Chemical compound, drug	Lucifer Yellow	Sigma	B4261	0.10%
Software, algorithm	SnapGene Viewer	SnapGene	RRID:SCR_015053
Software, algorithm	Ponemah	DSI	RRID:SCR_017107	Version 5.20
Software, algorithm	Spike	Cambridge Electronic Design	RRID:SCR_000903	Version 5.0
Software, algorithm	Sirenia	Pinnacile Technology	RRID:SCR_016183
Software, algorithm	Matlab	Mathworks	RRID:SCR_001622	Version R2018
Software, algorithm	Brainstorm	Tadel et al., 2011	RRID:SCR_001761	Version 3.0
Software, algorithm	Prism 7	GraphPad	RRID:SCR_002798	Version 7.03
Software, algorithm	pCLAMP 10	Molecular Devices	RRID:SCR_011323	Version 10
Software, algorithm	ImageJ	NIH	RRID:SCR_003070	Version 2.0.0

Table 3

Probes used for FISH.

https://doi.org/10.7554/eLife.43387.018

Gene name	Probe cat no.	Target region
Scn1a	434181	1624–2967
Slc32a1	319191-C2	894–2037
Slc17a6	319171-C2	1986–2998

Additional files

Transparent reporting form: https://doi.org/10.7554/eLife.43387.019
Download elife-43387-transrepform-v2.docx

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Figures

Conditional expression of Scn1a A1783V in inhibitory neurons results in premature death.

Figure 1—source data 1

Breeding strategy to generate mice that heterologously express the Scn1a A1783V pathological variant conditionally in inhibitory neurons (Scn1a^ΔE26 mice).

Scn1a^ΔE26 exhibit frequent spontaneous seizures.

Figure 2—source data 1

Scn1a^ΔE26 mice show reduced respiratory output under control conditions and during exposure to high CO₂.

Figure 3—source data 1

Brainstem inhibitory neurons in slices from Scn1a^ΔE26 show diminished basal activity and repetitive firing behavior during sustained depolarization.

Figure 4—source data 1

The Scn1a A1783V pathological variant may result in loss of channel function by increased voltage-dependent inactivation.

Figure 5—source data 1

Chemosensitive RTN neurons in slices from Scn1a^ΔE26 mice are hyper-excitable.

Figure 6—source data 1

Tables

Behavioral Assessment of Seizure activity.

Febrile seizure propensity.

Table 2—source data 1

Probes used for FISH.

Additional files

Transparent reporting form

Download links

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Cite this article

Conditional expression of Scn1a A1783V in inhibitory neurons results in premature death.

Figure 1—source data 1

Breeding strategy to generate mice that heterologously express the Scn1a A1783V pathological variant conditionally in inhibitory neurons (Scn1aΔE26 mice).

Scn1aΔE26 exhibit frequent spontaneous seizures.

Figure 2—source data 1

Scn1aΔE26 mice show reduced respiratory output under control conditions and during exposure to high CO2.

Figure 3—source data 1

Brainstem inhibitory neurons in slices from Scn1aΔE26 show diminished basal activity and repetitive firing behavior during sustained depolarization.

Figure 4—source data 1

The Scn1a A1783V pathological variant may result in loss of channel function by increased voltage-dependent inactivation.

Figure 5—source data 1

Chemosensitive RTN neurons in slices from Scn1aΔE26 mice are hyper-excitable.

Figure 6—source data 1

Behavioral Assessment of Seizure activity.

Febrile seizure propensity.

Table 2—source data 1

Probes used for FISH.

Transparent reporting form

Breeding strategy to generate mice that heterologously express the Scn1a A1783V pathological variant conditionally in inhibitory neurons (Scn1a^ΔE26 mice).

Scn1a^ΔE26 exhibit frequent spontaneous seizures.

Scn1a^ΔE26 mice show reduced respiratory output under control conditions and during exposure to high CO₂.

Brainstem inhibitory neurons in slices from Scn1a^ΔE26 show diminished basal activity and repetitive firing behavior during sustained depolarization.

Chemosensitive RTN neurons in slices from Scn1a^ΔE26 mice are hyper-excitable.