Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain

  1. Sophie L Fayad
  2. Guillaume Ourties
  3. Benjamin Le Gac
  4. Baptiste Jouffre
  5. Sylvain Lamoine
  6. Antoine Fruquière
  7. Sophie Laffray
  8. Laila Gasmi
  9. Bruno Cauli
  10. Christophe Mallet
  11. Emmanuel Bourinet
  12. Thomas Bessaih
  13. Régis C Lambert  Is a corresponding author
  14. Nathalie Leresche
  1. Sorbonne Université, CNRS, INSERM, Neurosciences Paris Seine – Institut de Biologie Paris Seine, France
  2. Université Clermont Auvergne, Inserm, U1107 Neuro-Dol, Pharmacologie Fondamentale et Clinique de la Douleur, France
  3. ANALGESIA Institute, Faculty of Medicine, France
  4. Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM, France
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Coexpression of Cav3.2 and parvalbumin (PV) in APT neurons.

Left panels: Cav3.2-GFP immunostaining on a parasagittal (A) and a coronal (B) section of KI mice brains. Right panels: corresponding Mouse Brain Atlas slides from Paxinos and Franklin (parasagittal: 1.08 mm lateral from Bregma; coronal: 2.8 mm posterior from Bregma). APT: anterior pretectum; Cx: cortex; Hpc: hippocampus; nRT: nucleus reticularis thalami; PAG: periaqueductal grey; SC: superior colliculi; SNc: substantia nigra pars compacta; ZI: zona incerta. (C) Confocal microscopy images of a coronal KI mouse brain section of the APT with GFP (green) and PV (red) co-labeling. Scale bar: 100 µm.

Figure 1—source data 1

Quantification of Cav3.2-GFP- and parvalbumin (PV)-expressing neurons.

https://cdn.elifesciences.org/articles/79018/elife-79018-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Anterior pretectum (APT) neurons expressing GFP and parvalbumin (PV).

Confocal microscopy images of the co-labelings performed in the APT to estimate the proportion of GFP neurons (A: GFP in green, NeuN in red) and PV neurons (B: PV in green, NeuN in red) and the overlap between these populations (C: GFP in green, PV in red). Scale bar: 30 µm. (D) Diagram representing the percentage of neurons expressing GFP and PV in the APT.

Figure 2 with 1 supplement
Impact of spared nerve injury (SNI) in PV+ anterior pretectum (APT) neurons.

(A) Example image of a DiI track left by a recording electrode inserted into the APT. Red: DiI. (B) Raw signals from the four wires of a tetrode. Bottom trace shows the simultaneous EEG recording. (C) Left panel: example of a tetrode recording where two units were isolated. All detected action potentials are plotted as their waveform’s amplitude from channel 1 versus the amplitude of their waveform’s valley from channel 1 (in arbitrary units, A.U.). Using this feature space arrangement, two single-unit clusters were isolated (in red and yellow). Right panel: superimposed color-coded action potential waveforms captured by each recording site of the tetrode are shown for the two identified single units (the polarity of the signals is inverted). (D) Example of peristimulus time histograms illustrating spiking response to optogenetic stimulation (10 ms long, represented in blue) over 100 trials of a unit categorized into the PV+ category. (E) Autocorrelogram of recorded single unit for one example cell. 1 ms bins were used. Red dotted lines represent 99% confidence intervals. (F) Scatter dot plots of mean firing rate (left panel), proportion of spikes within a burst (middle panel), and mean spike frequency within a burst (right panel) for fast-bursting APT cells recorded in sham (n = 21 cells, 5 animals) and SNI (n = 18 cells, 4 animals) mice. p values for statistical comparisons were obtained using Wilcoxon sum rank test.

Figure 2—source data 1

Firing properties of parvalbumin (PV)-expressing neurons after spared nerve injury (SNI).

https://cdn.elifesciences.org/articles/79018/elife-79018-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Anterior pretectum (APT) regular spiking neurons.

(A) Example of peristimulus time histograms illustrating spiking response to optogenetic stimulation (10 ms long, represented in blue) over 100 trials of a unit categorized into the PV+ category. (B) Autocorrelogram of recorded single unit for one example cells. 1 ms bins were used. Red dotted lines represent 99% confidence intervals.

Figure 2—figure supplement 1—source data 1

Firing properties of PV+ regular spiking neurons.

https://cdn.elifesciences.org/articles/79018/elife-79018-fig2-figsupp1-data1-v2.xlsx
PV+ neurons expressing Cav3.2 channels are GABAergic fast-spiking neurons.

(A) Left image: recorded neuron filled with biocytin (green). Neurons in red are nonrecorded PV+ neurons. Middle traces: depolarizing current injection evokes characteristic fast-spiking activity. Hyperpolarizing current injection evoked a pronounced sag and a rebound high-firing burst upon repolarization. Inset: enlargement of the bursting activity. Right graph: number of spikes evoked by 1 s long depolarizing current injection of increasing amplitudes. (B) Typical example of activities and scRT-PCR products observed in a PV+ neuron. Note the expression of the Cav3.2 channel in the GAD65- and 67-positive neuron.

Cav3.2 channel contribution to the bursting activity of PV+ neurons is enhanced in spared nerve injury (SNI) mice.

(A) Effect of 100 µM Ni2+ applications on the number of spikes (left graph, n = 8) and the minimal interspike intervals (ISIs; right graph, n = 6) of the rebound bursts. Typical examples of these pharmacological effects are shown on the right. (B) Effect of 1 µM TTAP2 applications on the number of spikes (left graph, n = 11) and the minimal ISIs (right graph, n = 7) of the rebound bursts. Typical examples of these pharmacological effects are shown on the right. (C) Effect of 100 µM Ni2+ (left graph, n = 10) and 1 µM TTAP2 (right graph, n = 12) applications on the amplitude of the rebound depolarization observed in the presence of 0.5 µM TTX. A typical example is presented in superimposed traces presented on the right. (D) The two left graphs compare the maximal number of spikes of the rebound bursts evoked in neurons of sham-operated and SNI mice in control condition (n = 12 and 15, respectively) and after application of 100 µM Ni2+ (n = 8 and 9, respectively). The effects of Ni2+ application on each neuron are presented in the two right graphs (n = 8 and 9 for sham and SNI, respectively). A, B, C, D right graphs: Wilcoxon signed rank test; D left graphs: Wilcoxon sum rank test. ***p < 0.001 ; **p: 0.001 < p < 0.01 ; *0.01 < p < 0.05.

Figure 4—source data 1

Ni and TTA effects on burst properties of PV+ neurons.

https://cdn.elifesciences.org/articles/79018/elife-79018-fig4-data1-v2.xlsx
Figure 4—source data 2

Ni and TTA effects on the rebound depolarization in PV+ neurons.

https://cdn.elifesciences.org/articles/79018/elife-79018-fig4-data2-v2.xlsx
Figure 4—source data 3

Burst properties of PV+ neurons in slices from sham and spared nerve injury (SNI) mice.

https://cdn.elifesciences.org/articles/79018/elife-79018-fig4-data3-v2.xlsx
Figure 5 with 1 supplement
Cav3.2 preventive and therapeutic knockout in the anterior pretectum (APT) alleviates neuropathy induced by spared nerve injury (SNI).

(A) Confocal microscopy images of GFP (green) and mCherry (red) co-labeling in the APT of KI-Cav3.2-GFP (Control) mice and KO-Cav3.2-APT (KO) bilaterally injected in the APT with AAV8-hSyn-mCherry and AAV8-hSyn-mCherry-CRE virus, respectively, and further tested for mechanical and cold sensitivity. Scale bar: 100 µm. Neuropathic behaviors were tested in male (dashed lines) and female (solid lines) mice with preventive (B, D) and therapeutic (C, E) KO of Cav3.2 in the APT (orange, ■), and in control KI mice (green, ●). (B, C) Mechanical sensitivity was assessed by measuring paw withdrawal thresholds (PWTs) in response to Von Frey filaments stimulations using the up-and-down method. (D, E) Cold sensitivity was assessed by measuring the paw withdrawal latency in response to immersion in 18°C water. For preventive KO (B, D) mice were tested prior to the SNI (BL) and once a week during the 6 subsequent weeks (days 14–48). For therapeutic KO (C, E) mechanical and cold sensitivity were assessed before (BL) and 14 days after SNI (SNI), and tested during several weeks following the subsequent viral injections (days 14–56 after viral injection). (B–E) Wilcoxon sum rank test for female KO versus KI and male KO versus KI comparison at each time point. ++++ : p < 0.0001, +++ or ▲▲▲: 0.0001 < p < 0.001, ++ or ▲▲: 0.001 < p < 0.01, + or ▲: 0.01 < p < 0.05 (males: +; females: ▲).

Figure 5—source data 1

Characterization of the effect of APT-Cav3.2 preventive or curative knockout on neuropathic pain induced by spared nerve injury (SNI).

https://cdn.elifesciences.org/articles/79018/elife-79018-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Preventive Cav3.2 knockout in the anterior pretectum (APT) has no impact on motor coordination and spontaneous locomotion.

(A) Confocal microscopy images of GFP labeling (green) and mCherry expression (red) in the APT of a KI mouse unilaterally injected with AAV8-hSyn-Cre-mCherry virus (up panel: injected hemisphere; down panel: noninjected hemisphere). Scale bar: 100 µm. Note the drastic reduction in the number of GFP+ neurons observed 2 weeks post viral injection in the injected hemisphere. (B–D) Motor coordination and spontaneous locomotion assessed in control KI mice (green, ●) and mice with preventive APT-Cav3.2 KO (orange, ■) subjected to spared nerve injury (SNI). (B) Control of the development of mechanical allodynia in male (dashed lines) and female (solid lines) mice. Note that as for the cohorts presented in Figure 5, preventive Cav3.2 KO reduced allodynia. Wilcoxon sum rank test for female KO versus KI and male KO versus KI comparison at each time point. +++ or ▲▲▲: 0.0001 < p < 0.001, ▲▲: 0.001 < p < 0.01, +: 0.01 < p < 0.05 (males: +; females: ▲). (C) Latency to fall from an accelerating rotarod wheel (0–16 rpm over 3 min) was measured in males (dashed bars) and females (solid bars), before (BL) and after (day 21) SNI surgery. No statistical differences were detected between groups and before and after SNI surgery within a group using Wilcoxon sum rank test and Wilcoxon signed rank test, respectively. (D) The number of quarter turns was detected in a circular corridor over 120 min, measured in males (dashed bars) and females (solid bars), 21 days after SNI surgery. No significant differences were detected between groups using the Wilcoxon sum rank test.

Figure 5—figure supplement 1—source data 1

Characterization of preventive APT-Cav3.2 knockout effect on mechanical sensitivity and locomotion.

https://cdn.elifesciences.org/articles/79018/elife-79018-fig5-figsupp1-data1-v2.xlsx

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(Mus musculus, male and female)
Cav3.2-eGFPfloxPMID:25600872
(François et al., 2015)
Cacna1htm1.1(epH)Ebo/JKI mice with two loxP site-flanked
ecliptic-GFP tag into exon 6 of
the Cacna1h gene
Strain, strain background
(Mus musculus, female)
PV-CreJackson labB6.129P2-
Pvalb-tm1(cre)Arbr/J
Cre recombinase expression
in Pvalb-expressing cells
Strain, strain background
(Mus musculus, male)
Ai14Jackson labB6.Cg-Gt(ROSA)
26Sor-tm14(CAG-tdTomato)Hze/J
Cre-dependent expression
of the red fluorescent protein
variant (tdTomato)
Strain, strain background
(Mus musculus, male)
Ai32Jackson labB6.Cg-Gt(ROSA)
26Sortm32(CAG-COP4*H134R/EYFP)Hze/J
Cre-dependent expression of the
ChR2(H134R)-EYFP
Sequence-based reagentMouse Pvalb external sensePMID:21795545
(Tricoire et al., 2011)
PCR primersGCCTGAAGAAAAAGAACCCG
Sequence-based reagentMouse Pvalb external antisensePMID:21795545
(Tricoire et al., 2011)
PCR primersAATCTTGCCGTCCCCATCCT
Sequence-based reagentMouse Pvalb internal sensePMID:21795545
(Tricoire et al., 2011)
PCR primersCGGATGAGGTGAAGAAGGTGT
Sequence-based reagentMouse Pvalb internal antisensePMID:21795545
(Tricoire et al., 2011)
PCR primersTCCCCATCCTTGTCTCCAGC
Sequence-based reagentMouse Gad2 external sensePMID:34766906
(Karagiannis et al., 2021)
PCR primersCCAAAAGTTCACGGGCGG
Sequence-based reagentMouse Gad2 external antisensePMID:34766906
(Karagiannis et al., 2021)
PCR primersGTGAGCAGTATCGCAGCCCC
Sequence-based reagentMouse Gad2 internal sensePMID:34766906
(Karagiannis et al., 2021)
PCR primersCACCTGCGACCAAAAACCCT
Sequence-based reagentMouse Gad2 internal antisensePMID:12196560
(Férézou et al., 2002)
PCR primersGATTTTGCGGTTGGTCTGCC
Sequence-based reagentMouse Gad1 external sensePMID:23565079
(Cabezas et al., 2013)
PCR primersTACGGGGTTCGCA
CAGGTC CGGGCGG
Sequence-based reagentMouse Gad1 external antisensePMID:23565079
(Cabezas et al., 2013)
PCR primersCCCAGGCAGCATCCACAT
Sequence-based reagentMouse Gad1 internal sensePMID:23565079
(Cabezas et al., 2013)
PCR primersCCCAGAAGTGAAGACAAAAGGC
Sequence-based reagentMouse Gad1 internal antisensePMID:23565079
(Cabezas et al., 2013)
PCR primersAATGCTCCGTAAACAGTCGTGC
Sequence-based reagentMouse Slc17a6 external senseThis paperPCR primersTGGAGAAGAAGCAGGACAACC
Sequence-based reagentMouse Slc17a6 external antisenseThis paperPCR primersGTGAGCAGTATCGCAGCCCC
Sequence-based reagentMouse Slc17a6 internal senseThis paperPCR primersTGACAGAGGACGGTAAGCCCC
Sequence-based reagentMouse Slc17a6 internal antisenseThis paperPCR primersTCATCCCCACGGTCTCGG
Sequence-based reagentMouse Cacna1g external senseThis paperPCR primersCACCGATGTCACTGCCCAAG
Sequence-based reagentMouse Cacna1g external antisenseThis paperPCR primersGGCTCTCCTGACCCTCTCCA
Sequence-based reagentMouse Cacna1g internal senseThis paperPCR primersGCTCTCGCCGCACCAGTA
Sequence-based reagentMouse Cacna1g internal antisenseThis paperPCR primersCTTGGGCTCCTACGCTTCAG
Sequence-based reagentMouse Cacna1h external senseThis paperPCR primersTACCAGACAGAGGAGGGCGA
Sequence-based reagentMouse Cacna1h external antisenseThis paperPCR primersCTATCACCACCAGGCACAGG
Sequence-based reagentMouse Cacna1h internal senseThis paperPCR primersCATTCATCTGCTCCTCACGC
Sequence-based reagentMouse Cacna1h internal antisenseThis paperPCR primersGCCCACAATGATGAGGAGGA
Sequence-based reagentMouse Cacna1i external senseThis paperPCR primersGTCCCCCTCCATCCCCTC
Sequence-based reagentMouse Cacna1i external antisenseThis paperPCR primersCAATGAAGAAGTCCAAGCGGTT
Sequence-based reagentMouse Cacna1i internal senseThis paperPCR primersGTTGCCTTCTTCTGCCTGCG
Sequence-based reagentMouse Cacna1i internal antisenseThis paperPCR primersTCCCCGAGGTAGCACTTCTT
Strain, strain background (AAV)AAV8-hSyn-mCherry-CreUNC Vector Core
Strain, strain background (AAV)AAV8-hSyn-mCherryUNC Vector Core
AntibodyAnti-GFP
(chicken polyclonal)
Life TechnologiesA102621:500
AntibodyAnti-GFP
(rabit polyclonal)
ChromotekPABG11:500
AntibodyAnti-Parvalbumin (mouse monoclonal)Sigma-AldrichP30881:1000
AntibodyAnti-NeuN (rabbit polyclonal)Merck MilliporeABN781:500
AntibodyAnti-chicken-Alexa Fluor 488 (goat polyclonal)Life TechnologiesA110391:500
AntibodyAnti-chicken-Alexa Fluor 488 (donkey polyclonal)Sigma-AldrichSAB46000311:500
AntibodyAnti-rabit-Alexa Fluor 647 (goat polyclonal)Life TechnologiesA212441:500
AntibodyAnti-mouse-Alexa Fluor 488 (goat polyclonal)Life TechnologiesA110011:500
AntibodyAnti-rabit-Cyanine Cy3 (donkey polyclonal)Jackson ImmunoResearch
Europe
711-165-15251:2000
Chemical compound, drugIsofluraneIso-VetCat# 3248850;
GTIN: 18904026625157
Chemical compound, drugBuprenorphine (Buprecare)AxienceGTIN: 03760087151893
Chemical compound, drugPentobarbital (Euthasol Vet)DechraGTIN: 08718469445110
Chemical compound, drugKetamineImalgene 1000GTIN: 03661103003199
Chemical compound, drugXylazine (Rompun)BayerGTIN: 04007221032311
Chemical compound, drugBupivacaineHenry ScheinCat# 054879
Chemical compound, drugTwelve TVM Eye Support DropsTVM UK Animal HealthGTIN: 03700454507502
Chemical compound, drugVetedineVetoquinolGTIN: 03605870001385
Chemical compound, drugNaClSigma-AldrichCat# S6191;
CAS: 7647-14-5
Chemical compound, drugKClSigma-AldrichCat# 60128;
CAS: 7447-40-7
Chemical compound, drugCaCl2Sigma-AldrichCat# C3881;
CAS: 10035-04-8
Chemical compound, drugMgCl2Sigma-AldrichCat# M2670;
CAS: 7791-18-6
Chemical compound, drugNaH2PO4Sigma-AldrichCat# S5011;
CAS: 7558-80-7
Chemical compound, drugNaHCO3Sigma-AldrichCat# 31437;
CAS: 144-55-8
Chemical compound, drugGlucoseSigma-AldrichCat# 49159;
CAS: 14431-43-7
Chemical compound, drugPotassium gluconateSigma-AldrichCat# G4500;
CAS: 299-27-4
Chemical compound, drugEGTASigma-AldrichCat# E3889;
CAS: 67-42-5
Chemical compound, drugHEPESSigma-AldrichCat# H3375;
CAS: 7365-45-9
Chemical compound, drugDisodium ATPSigma-AldrichCat# A7699;
CAS: 34369-07-8
Chemical compound, drugBiocytinSigma-AldrichCat# B4261;
CAS: 576-19-2
Chemical compound, drugD-MannitolSigma-AldrichCat# M9647;
CAS: 69-65-8
Chemical compound, drugSodium PyruvateSigma-AldrichCat# P2256;
CAS: 113-24-6
Chemical compound, drugKynurenic acidHello BioCat# HB0363;
CAS: 2439-02-3
Chemical compound, drugParaformaldehyde in 0.1 M phosphate buffer salineElectron Microscopy SciencesCat# 15710;
CAS: 30525-89-4
Chemical compound, drugTrizmaSigma-AldrichCat# T1503;
CAS: 77-86-1
Chemical compound, drugSodium azideSigma-AldrichCat# S2002;
CAS: 26628-22-8
Chemical compound, drugPhosphate buffer salineSigma-AldrichCat# D1408
Chemical compound, drugTriton X-100Sigma-AldrichCat# T8787;
CAS: 9002-93-1
Chemical compound, drugFluoromount Aquaous Mounting MediumSigma-AldrichCat# F4680
Chemical compound, drugDonkey serumSigma-AldrichCat# D9663
Chemical compound, drugStreptavidin-
Rhodamine-RedX
Jackson ImmunoResearch
Europe
Cat# 016-290-084
Chemical compound, drugDiI, 10% in ethanolInvitrogenCat# V22885
Chemical compound, drugCNQXHello bioCat# HB02057; CAS: 479347-85-8
Chemical compound, drugSR95531 hydrobromideHello bioCat# HB0901;
CAS: 104104-50-9
Chemical compound, drugTTXLatoxanCat# L8503;
CAS: 4368-28-9
Chemical compound, drugNickel chloride hydrateSigma-AldrichCat# 364304;
CAS: 69098-15-3
Chemical compound, drugTTA-P2Alomone labsCat# T-155;
CAS: 918430-49-6
Chemical compound, drugTaq DNA PolymeraseQiagen201205
Chemical compound, drugRNasin Ribonuclease InhibitorsPromegaN2511
Chemical compound, drugSuperScript II Reverse TranscriptaseInvitrogenCat# 18064014
Software, algorithmPclamp V 10.2Molecular Deviceshttps://www.moleculardevices.com/
Software, algorithmMatlab 2019bMathWorkshttps://fr.mathworks.com/
Software, algorithmIgor Pro V6Wavemetricshttps://www.wavemetrics.com
Software, algorithmCheetah V 5Neuralynxhttps://neuralynx.com/software/cheetah
Software, algorithmKlustaKwikNeuralynxhttps://neuralynx.com/software/cheetah
Software, algorithmSpikeSort3D V 2Neuralynxhttps://neuralynx.com/software/cheetah
Software, algorithmFiji/ImageJNIHhttps://imagej.nih.gov/ij/download.html
Software, algorithmRstudiohttps://www.rstudio.com/
Software, algorithmR version 4.1.0 (2021-05-18)Camp Pontanezen – The R Foundation for Statistical Computinghttps://www.r-project.org/
OtherQuartz-insulated platinum/tungsten (90%/10%) tetrodesThomas Recording GmbHCat# AN000259See ‘In vivo electrophysiological
recordings’ in the Method Details
OtherOptical fibersThomas Recording GmbHCat# AN000514Optical fiber used to deliver 470-nm
blue-light pulse for Photo-assisted
Identification of Neuronal Population
OtherBorosilicate glass capillariesHilgenbergCat# 1409250See ‘In vitro whole-cell patch-clamp
recording’ in the Method Details
Other4-0 VicrylEthiconref JV397See ‘Virus stereotaxic injections’ in
the Method Details

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  1. Sophie L Fayad
  2. Guillaume Ourties
  3. Benjamin Le Gac
  4. Baptiste Jouffre
  5. Sylvain Lamoine
  6. Antoine Fruquière
  7. Sophie Laffray
  8. Laila Gasmi
  9. Bruno Cauli
  10. Christophe Mallet
  11. Emmanuel Bourinet
  12. Thomas Bessaih
  13. Régis C Lambert
  14. Nathalie Leresche
(2022)
Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain
eLife 11:e79018.
https://doi.org/10.7554/eLife.79018