Figures and data in Calpain fosters the hyperexcitability of motoneurons after spinal cord injury and leads to spasticity

Version of Record: December 23, 2019
Accepted Manuscript: December 9, 2019

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Vanessa Plantier

Irene Sanchez-Brualla

Nejada Dingu

Cécile Brocard

Sylvie Liabeuf

Florian Gackière

Frédéric Brocard

(2019)
Calpain fosters the hyperexcitability of motoneurons after spinal cord injury and leads to spasticity

eLife 8:e51404.

https://doi.org/10.7554/eLife.51404
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Figures
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Additional files

6 figures, 1 table and 1 additional file

Figures

Figure 1 with 5 supplements

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Calpain inhibition alleviates early behavioral signs of spasticity in neonatal rats with SCI.

(**A,B**) Pictures of typical hindlimb motor response to tail pinching in sham-operated (A), n = 6 rats) and SCI rats (B), 5 d post-SCI, n = 8 rats). Lower and upper traces represent the pinch force and …

Figure 1—source data 1 Values displayed in bar plots in Figure 1C-G.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig1-data1-v2.xlsx
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Figure 1—figure supplement 1

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Calpain inhibition does not affect the number of twitches recorded in intact neonatal rats.

Time-course changes of twitches after acute i.p. administration (t = 0) of MDL28170 (120 mg/kg) in intact neonatal rats (4–5 d old; n = 6 rats). n.s. p>0.05, repeated measures one-way ANOVA, …

Figure 1—figure supplement 1—source data 1 Values displayed in the time course shown in Figure 1—figure supplement 1.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig1-figsupp1-data1-v2.xlsx
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Figure 1—video 1

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posterframe for video — Typical hindlimb motor movements to tail pinching in sham-operated neonatal rats.

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Figure 2

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The expression of Calpain-I increased after SCI.

*On the left*, calpain-I and calpain-II immunoblots of lumbar segments in sham-operated (n = 4 rats) and SCI neonatal rats (5 d post-SCI, n = 4 rats). One rat per lane. *On the right*, group means …

Figure 2—source data 1 Values displayed in bar plots in Figure 2.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig2-data1-v2.xlsx
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Figure 3 with 1 supplement

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Early development of hyperexcitability on caudal spinal cord after SCI.

(**A–C, J–L**) Representative L5 ventral root activities (VR L5) evoked by a supramaximal stimulation (St.) of the ipsilateral dorsal root (**A–C**) or occurring spontaneously (**J–L**) in spinal cords isolated …

Figure 3—source data 1 Values displayed in bar plots shown in Figure 3G-I, M-O.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig3-data1-v2.xlsx
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Figure 3—figure supplement 1

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Stability of both hyperreflexia and spontaneous activities in spinal cord isolated from neonatal rats with SCI.

(A) Superimposition of the first (black) and the last (red) dorsal root evoked L5 ventral root responses recorded in spinal cord isolated from SCI rats (4–5 d post-SCI) during a stimulation session …

Figure 3—figure supplement 1—source data 1 Values displayed in bar plots shown in Figure 3—figure supplement 1B–D, F.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig3-figsupp1-data1-v2.xlsx
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Figure 4 with 2 supplements

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Inhibition of calpain restores the excitatory/inhibitory equilibrium of motoneurons.

(**A,D**) Representative L5 ventral root activities (VR L5) evoked by a supramaximal stimulation (St.) of the ipsilateral dorsal root (A) or occurring spontaneously (D) in spinal cords isolated from SCI …

Figure 4—source data 1 Values displayed in Figure 4C, E-K.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig4-data1-v2.xlsx
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Figure 4—figure supplement 1

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Calpain inhibition affects neither I_NaP nor E_IPSP in lumbar motoneurons recorded from neonatal intact rats.

(A) Leak-subtracted I_NaP in lumbar motoneurons (n = 6 cells) from intact rats (5 d old) before (black) and after (red) bath-applying MDL28170 (30 µM, 30–60 min). (B) Quantification of I_NaP. n.s. p>0.…

Figure 4—figure supplement 1—source data 1 Values displayed in plots shown in Figure 4—figure supplement 1B, D.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 2

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Cleavage of KCC2 channels by calpain-1.

(A) KCC2 immunoblot from intact neonatal rat with incremental concentration of calpain-1 (B). Quantification of ~240 kDa and ~140 kDa bands in calpain-treated homogenates, normalized to untreated …

Figure 4—figure supplement 2—source data 1 Values displayed in bar plots shown in Figure 4—figure supplement 2B, D.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig4-figsupp2-data1-v2.xlsx
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Figure 5 with 2 supplements

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*I_NaP*-blockers or a KCC2-enhancer normalize the excitability of the spinal cord below SCI.

(**A,D,F,I,K,N**) Representative L5 ventral root activities (VR L5) evoked by a supramaximal stimulation (St.) of the ipsilateral dorsal root (**A,F,K**) or occurring spontaneously (**D,I,N**) in spinal cords …

Figure 5—source data 1 Values displayed in bar plots shown in Figure 5C, E, H, J, M, O.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig5-data1-v2.xlsx
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Figure 5—figure supplement 1

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The I_NaP-enhancer veratridine used at 100 nM triggers riluzole-sensitive hyperreflexia and spontaneous activities in isolated spinal cords from intact neonatal rats.

(**A,D**) Representative L5 ventral root activities (VR L5) evoked by a supramaximal stimulation (St.) of the ipsilateral dorsal root (A) or occurring spontaneously (D) in spinal cords isolated from …

Figure 5—figure supplement 1—source data 1 Values displayed in bar plots shown in Figure 5—figure supplement 1C, E.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig5-figsupp1-data1-v2.xlsx
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Figure 5—figure supplement 2

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I_CaP does not contribute to hyperexcitability caudal to SCI.

(**A,D,F,I**) Representative L5 ventral root activities (VR L5) evoked by a supramaximal stimulation (St.) of the ipsilateral dorsal root (**A,F**) or occurring spontaneously (**D,I**) before (black) and after …

Figure 5—figure supplement 2—source data 1 Values displayed in bar plots shown in Figure 5—figure supplement 2C, E, H, J.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig5-figsupp2-data1-v2.xlsx
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Figure 6

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E_IPSP and I_NaP act synergistically to promote spinal hyperexcitability.

(A) Representative L5 ventral root activities (VR L5) evoked by a supramaximal stimulation (St.) of the ipsilateral dorsal root in spinal cords isolated from intact rats (4–5 d old, n = 11 rats) …

Figure 6—source data 1 Values displayed in bar plots shown in Figure 6C, F, H.: https://cdn.elifesciences.org/articles/51404/elife-51404-fig6-data1-v2.xlsx
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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	mouse monoclonal anti-PanNav	Sigma	clone K58/35 CAT#S8809 RRID:AB_477552	(1:500)
Antibody	rabbit polyclonal anti-Na_v1.6	Alomone	Cat # ASC-009 RRID:AB_2040202	(1:200)
Antibody	rabbit polyclonal anti-KCC2	Millipore	CAT# 07–432 RRID:AB_310611	(1:400, 1:500)
Antibody	rabbit polyclonal anti-calpain-I	Ozyme	CAT#2556S	(1:500)
Antibody	rabbit polyclonal anti-calpain-II	Millipore	CAT#AB81023 RRID:AB_1586917	(1:500)
Antibody	ImmunoPure goat HRP-conjugated mouse-specific antibody	Thermo Fisher Scientific	CAT#0031430 RRID:AB_228307	(1:40000)
Antibody	AlexaFluor-488 goat anti-mouse IgG2b	Thermo Fisher Scientific	CAT# A-21141 RRID:AB_141626	(1:800)
Antibody	AlexaFluor-546 F(ab’)two goat anti-rabbit IgG	Thermo Fisher Scientific	CAT# A-11071 RRID:AB_2534115	(1:400)
Chemical compound, drug	NaCl	Sigma-Aldrich	CAT# 71376
Chemical compound, drug	KCl	Sigma-Aldrich	CAT# P3911
Chemical compound, drug	NaH₂PO₄	Sigma-Aldrich	CAT# S0751
Chemical compound, drug	MgSO₄	Sigma-Aldrich	CAT# 1880
Chemical compound, drug	CaCl₂	Sigma-Aldrich	CAT# 21115
Chemical compound, drug	NaHCO₃	Sigma-Aldrich	CAT# S6014
Chemical compound, drug	D-glucose	Sigma-Aldrich	CAT# G8270
Chemical compound, drug	K⁺-gluconate	Sigma-Aldrich	CAT# P1847
Chemical compound, drug	MgCl₂	Sigma-Aldrich	CAT# M8266
Chemical compound, drug	HEPES	Sigma-Aldrich	CAT# H3375
Chemical compound, drug	EGTA	Sigma-Aldrich	CAT# E3889
Chemical compound, drug	ATP	Sigma-Aldrich	CAT# A9062
Chemical compound, drug	GTP	Sigma-Aldrich	CAT# G9002
Chemical compound, drug	Sucrose	Sigma-Aldrich	CAT# S9378
Chemical compound, drug	Cadmium Chloride	Sigma-Aldrich	CAT# 202908
Chemical compound, drug	Tetraethylammonium chloride	Sigma-Aldrich	CAT# 86616
Chemical compound, drug	Nifedipine	Sigma-Aldrich	CAT# N7634
Chemical compound, drug	Dimethylsulphoxide	Sigma-Aldrich	CAT# D8418
Chemical compound, drug	Paraformaldehyde	EMS	CAT# 15714 s
Chemical compound, drug	Phosphate Buffered Saline	Argene Biomérieux	CAT# 33–011
Chemical compound, drug	Tissue-Tek OCT compound	VWR	CAT# 25608–930
Chemical compound, drug	Tris-buffered saline	Bio-world	CAT# 105300272
Chemical compound, drug	Triton X-100	Sigma-Aldrich	CAT# T9284
Chemical compound, drug	CompleteMini	Roche diagnostic Basel	CAT#11836170001
Chemical compound, drug	Iodoacetamide	Sigma-Aldrich	CAT#I1149
Chemical compound, drug	Igepal CA-630	Sigma-Aldrich	CAT#I8896
Chemical compound, drug	SDS	Sigma-Aldrich	CAT#05030–1 L-F
chemical compound, drug	Acryl/Bisacrylamide solution	Biorad	CAT#161–0146
Chemical compound, drug	PMSF	Sigma-Aldrich	CAT#93482
Chemical compound, drug	Pepstatin A	Sigma-Aldrich	CAT#77170
Chemical compound, drug	MDL28170	Calbiochem	CAT#208722
Chemical compound, drug	Calpain 1	Calbiochem	CAT#208712
Chemical compound, drug	Bay K8644	Sigma-Aldrich	CAT# B112
chemical compound, drug	CNQX	Sigma-Aldrich	CAT# C127
Chemical compound, drug	DIOA	Sigma-Aldrich	CAT# D129
Chemical compound, drug	Veratridine	Sigma-Aldrich	CAT# V5754
Chemical compound, drug	AP5	Tocris	CAT# 3693/10
Chemical compound, drug	PCPz	Tocris	CAT# 3287/100
Chemical compound, drug	Riluzole	Tocris	CAT# 0768/25
Chemical compound, drug	4,9-ah-TTX	Focus Biomolecules	CAT# 10–3700
Strain, strain background (Rattus norvegicus)	Wistar rats	Charles River Laboratories	RRID:RGD_2308816
Software, algorithm	pClamp v10.3	Molecular Devices	RRID:SCR_011323
Software, algorithm	ImageJ v1.50i	https://imagej.nih.gov/ij/	RRID:SCR_003070
Software, algorithm	FluoView v5.0	Olympus	RRID:SCR_014215
Software, algorithm	Image Lab v5.1	Bio-Rad	RRID:SCR_014210
Software, algorithm	Graphpad Prism	Prism	RRID:SCR_002798
Other	Vibrating microtome	Leica	VT1000S RRID:SCR_016495
Other	Temperature controller	Warner Instruments	CL-100
Other	Nikon Eclipse microscope	Nikon	E600FN
Other	Confocal microscope	Zeiss	LSM510
Other	Infrared-sensitive CCD camera	Hitachi	KP-200/201
Other	Digidata 1440a interface	Molecular Devices	N/A
Other	Multiclamp 700B amplifier	Molecular Devices	N/A
Other	Borosilicate glass capillaries	World Precision Instruments	CAT# TW150-4
Other	Sutter P-97 puller	Sutter Instruments	RRID:SCR_016842
Other	Knittel Glass coverslips	Dutscher	CAT# 900529
Other	Polysine slides	Thermoscientific	CAT# P4981
Other	Small Animal Ventilator	CWE	SAR-830/AP
Other	Cardiotachometer	CWE	CT-1000
Other	Chemidoc imaging system XRS+	Bio-rad	RRID:SCR_014210

Additional files

Transparent reporting form: https://cdn.elifesciences.org/articles/51404/elife-51404-transrepform-v2.docx
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Downloads (link to download the article as PDF)

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Figures

Calpain inhibition alleviates early behavioral signs of spasticity in neonatal rats with SCI.

Figure 1—source data 1

Calpain inhibition does not affect the number of twitches recorded in intact neonatal rats.

Figure 1—figure supplement 1—source data 1

Typical hindlimb motor movements to tail pinching in sham-operated neonatal rats.

Typical hindlimb motor movements to tail pinching in spinal cord transected neonatal rats.

Typical spontaneous tail and hindlimb movements in sham-operated neonatal rats.

Typical spontaneous tail and hindlimb movements in spinal cord transected neonatal rats.

The expression of Calpain-I increased after SCI.

Figure 2—source data 1

Early development of hyperexcitability on caudal spinal cord after SCI.

Figure 3—source data 1

Stability of both hyperreflexia and spontaneous activities in spinal cord isolated from neonatal rats with SCI.

Figure 3—figure supplement 1—source data 1

Inhibition of calpain restores the excitatory/inhibitory equilibrium of motoneurons.

Figure 4—source data 1

Calpain inhibition affects neither I_NaP nor E_IPSP in lumbar motoneurons recorded from neonatal intact rats.

Figure 4—figure supplement 1—source data 1

Cleavage of KCC2 channels by calpain-1.

Figure 4—figure supplement 2—source data 1

I_NaP-blockers or a KCC2-enhancer normalize the excitability of the spinal cord below SCI.

Figure 5—source data 1

The I_NaP-enhancer veratridine used at 100 nM triggers riluzole-sensitive hyperreflexia and spontaneous activities in isolated spinal cords from intact neonatal rats.

Figure 5—figure supplement 1—source data 1

I_CaP does not contribute to hyperexcitability caudal to SCI.

Figure 5—figure supplement 2—source data 1

E_IPSP and I_NaP act synergistically to promote spinal hyperexcitability.

Figure 6—source data 1

Tables

Additional files

Transparent reporting form

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Cite this article

Calpain inhibition alleviates early behavioral signs of spasticity in neonatal rats with SCI.

Figure 1—source data 1

Calpain inhibition does not affect the number of twitches recorded in intact neonatal rats.

Figure 1—figure supplement 1—source data 1

Typical hindlimb motor movements to tail pinching in sham-operated neonatal rats.

Typical hindlimb motor movements to tail pinching in spinal cord transected neonatal rats.

Typical spontaneous tail and hindlimb movements in sham-operated neonatal rats.

Typical spontaneous tail and hindlimb movements in spinal cord transected neonatal rats.

The expression of Calpain-I increased after SCI.

Figure 2—source data 1

Early development of hyperexcitability on caudal spinal cord after SCI.

Figure 3—source data 1

Stability of both hyperreflexia and spontaneous activities in spinal cord isolated from neonatal rats with SCI.

Figure 3—figure supplement 1—source data 1

Inhibition of calpain restores the excitatory/inhibitory equilibrium of motoneurons.

Figure 4—source data 1

Calpain inhibition affects neither INaP nor EIPSP in lumbar motoneurons recorded from neonatal intact rats.

Figure 4—figure supplement 1—source data 1

Cleavage of KCC2 channels by calpain-1.

Figure 4—figure supplement 2—source data 1

INaP-blockers or a KCC2-enhancer normalize the excitability of the spinal cord below SCI.

Figure 5—source data 1

The INaP-enhancer veratridine used at 100 nM triggers riluzole-sensitive hyperreflexia and spontaneous activities in isolated spinal cords from intact neonatal rats.

Figure 5—figure supplement 1—source data 1

ICaP does not contribute to hyperexcitability caudal to SCI.

Figure 5—figure supplement 2—source data 1

EIPSP and INaP act synergistically to promote spinal hyperexcitability.

Figure 6—source data 1

Transparent reporting form

Calpain inhibition affects neither I_NaP nor E_IPSP in lumbar motoneurons recorded from neonatal intact rats.

I_NaP-blockers or a KCC2-enhancer normalize the excitability of the spinal cord below SCI.

The I_NaP-enhancer veratridine used at 100 nM triggers riluzole-sensitive hyperreflexia and spontaneous activities in isolated spinal cords from intact neonatal rats.

I_CaP does not contribute to hyperexcitability caudal to SCI.

E_IPSP and I_NaP act synergistically to promote spinal hyperexcitability.