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
  14. Nathalie Leresche

  • Curated by eLife

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    Evaluation Summary:

    The manuscript shows an important role for Cav2.3 channels in SNI-mediated allodynia and firing properties of PV-expressing APT neurons. Mechanisms that underlie adaptations in chronic pain models are extremely important for the development of novel therapeutics for chronic pain and this could be a significant contribution in that regard. However, the discussion asserts that these studies are the "first direct evidence that supra-spinal Cav3.2 channels play a fundamental role in pain pathophysiology." This is an overstatement as Chen and colleagues examined the role of these channels in the anterior cingulate cortex in CCI-mediated neuropathic pain (Shen, et al., 2015, Molecular Pain).

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Cav3.2 T-type calcium channel is a major molecular actor of neuropathic pain in peripheral sensory neurons, but its involvement at the supraspinal level is almost unknown. In the anterior pretectum (APT), a hub of connectivity of the somatosensory system involved in pain perception, we show that Cav3.2 channels are expressed in a subpopulation of GABAergic neurons coexpressing parvalbumin (PV). In these PV-expressing neurons, Cav3.2 channels contribute to a high-frequency-bursting activity, which is increased in the spared nerve injury model of neuropathy. Specific deletion of Cav3.2 channels in APT neurons reduced both the initiation and maintenance of mechanical and cold allodynia. These data are a direct demonstration that centrally expressed Cav3.2 channels also play a fundamental role in pain pathophysiology.

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  1. Version published to 10.7554/elife.79018 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The manuscript "Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain" identifies a subset of parvalbumin-expressing GABAergic neurons in the anterior pretectum (APT) that co-express Cav2.3 T-type calcium channels. The firing frequency and burst patterns are potentiated in these neurons following spared nerve injury (SNI) and the development of neuropathic pain. Deletion of the channels in these cells reduced both the development and maintenance of mechanical and cold allodynia. Studies show nice co-expression of the PV and GFP in the Cav2.3.2eGFP-flox KI mouse line.

    Multi-unit recordings from PV-Cre X Ai32 mice show that PV neurons in the APT are fast-spiking and that the mean firing rate and frequency of spikes in bursts are potentiated in SNI animals. The graphs in Fig. 2, panel F show compiled data of 18-20 cells from 6-8 animals depending on the treatment. The statistical design for the in vivo experiments (and actually all of the studies) are not clearly stated with degrees of freedom. It is important to know if recordings from a single animal are considered independent observations, and if so, what the rationale for that is. This information should be included in the Quantification and Statistical Analysis section. In addition, it would be interesting to determine if T-type calcium channel blockers can reverse this behavior in these recordings.

    We considered each unit as an independent observation. We did so since the number of recorded PV+-units per animal (identified with the PINP method) was small and varied greatly between animals, from 2 to 6 units. We are not aware of statistical methods using a nested design for multiple cells in animals that could be used in such condition.

    Since the measured variables did not follow normal distributions, we performed unpaired comparison with the Wilcoxon sum rank test. This is now stated in the section ‘quantification and statistical analysis’ (page 19, line 683). P-values are now included in the figures and the result section when appropriate.

    In vitro electrophysiological studies show that the PV-expressing APT neurons exhibit fast-spiking to depolarization and single-cell RT-PCR shows that Cav3.2 is expressed in APT neurons that also express GABA. These cells show an after-hyperpolarization burst of APs that is reduced by blockers of Cav3.2 channels. There are no statistics displayed on panels C-E in Fig. 3, although they are reported in the text. Again, the test used and degrees of freedom, etc. should also be reported as it allows for evaluation of the experimental design.

    We apologize for the lack of statistics in Fig. 3 (now Fig. 4). Statistics are now clearly presented on each figure panel and the statistical tests are stated in the figure legends and in the results (page 4, line 144-159).

    As it is now stated in the “Quantification and Analysis section” (page 19, line 682), each neuron was considered as an independent observation since 1 to 3 neurons were recorded per mouse. The number of mice and the mean number of neurons per mouse are indicated in the data-set for each experimental condition in order to allow for a clear evaluation of the experimental design. Note that in the experiments with application of T-channel antagonist, only one neuron was recorded per slice. This is now specified in the Method Details (page 17, line 579).

    It is also noted in the Discussion (lines 185-186) that "Our in vitro data indicate that 92% of APT-PV+ neurons are able to discharge bursts of action potentials at high frequency underpinned by a large transient depolarization due to the activation of T channels." It would be more clear to refer to the rebound as the figure also shows the fast-spiking properties due to depolarization as well as the transient depolarization due to the rebound but only an effect of the Cav2.3 on the rebound.

    We agree and have changed the sentence accordingly (page 5, line 202).

    Behavioral studies of mechanical and cold allodynia in male and female naïve and SNI-treated KI and KO mice were performed. These results show a clear contribution of the Cav3.2 channels in APT in both the development and maintenance of neuropathic pain. Again, the statistical design is not clearly defined and it is extremely difficult to resolve what comparisons are delineated in panels B-E of Fig. 4.

    We fully agree with the reviewer that the rationale for the choice of statistical tests used to analyze the behavioral data was lacking. We have rewritten the relevant paragraph in the Quantification and Analysis section (page 19, lines 699-714). The statistical results presented in the Fig 4 and its supplemental figure (now Fig 5 and Figure 5 – Figure supplement 1) are now clearly stated in the legends.

    Reviewer #3 (Public Review):

    The authors used state-of-the-art techniques to investigate the role of centrally located (GABAergic APT neurons) CaV3.2 isoform of T-channels in an animal model of neuropathic pain using speared nerve injury model. This is generally an excellent and very rigorous study. The data is very compelling and it is likely going to have a major impact in the field of ion channels and pain transmission. The data presentation is superb and major conclusions are highly justified. Major strengths include the use of powerful complementary techniques such as molecular (single-cell PCR), mouse genetics, and pain testing in vivo, as well as sophisticated ex vivo (slice physiology) and in vivo recordings (burst analysis using tetrodes). This study may explain recent clinical studies that failed to show the efficacy of peripherally acting Cav3.2 channel blockers in patients with neuropathic pain. Hence, this study has the potential to change the focus from peripheral to supraspinal Cav3.2 channels in various pain pathologies.

    Some moderate weaknesses are identified and should be addressed:

    1. The data showing the effect of T-channel deletion on the excitability of GABAergic neurons of APT is very convincing. However, what is missing is a discussion of how changes in the excitability of inhibitory APT neurons impact the circuitry that is involved. Without knowing the circuitry involved, one could speculate that blocking inhibitory drive may do just the opposite effect of what is proposed and increase hyperalgesia.

    We agree with the reviewer that discussing this issue is essential and it has now been added (page 6, lines 270-295).

    1. Methods should clearly state if any experiments were done in a blinded fashion.

    Behavioral experiments were performed blind. This has been added in the method section (page 18, line 624). For in vivo electrophysiological experiments, we cannot say that we performed blind experiments (although we tried). Indeed, under anesthesia, the forelimb of SNI animals presents a slight but observable withdrawal.

    1. There is no mention anywhere of how was selective Cav3.2 knock-out achieved, nor how was this assessed. It would be very helpful if authors could perform recordings of T-channel amplitudes in sham animals, animals after SNI and after selective knock-out in the SNI group.

    The efficiency of the Cav3.2 deletion after Cre virus injection was assessed by immunolabeling of GFP in APT slices. As shown in Figure 5 – Figure supplement 1, we checked that unilateral injection of AAV8-hSyn-Cre-mCherry virus induced a drastic reduction in the number of GFP+ neurons when compared to the non-injected hemisphere. The absence of Cav3.2 expression in Cre injected APTs was systematically checked in each mouse at the end of the behavioral tests (Figure 5A). This is now added in the Method Details (page 18, line 655).

    1. It should be discussed that global Cav3.2 animals had only minimal neuropathic pain phenotype (Choi et al., 2007).

    This point is now discussed (page 6, line 251).

  3. eLife

    Evaluation Summary:

    The manuscript shows an important role for Cav2.3 channels in SNI-mediated allodynia and firing properties of PV-expressing APT neurons. Mechanisms that underlie adaptations in chronic pain models are extremely important for the development of novel therapeutics for chronic pain and this could be a significant contribution in that regard. However, the discussion asserts that these studies are the "first direct evidence that supra-spinal Cav3.2 channels play a fundamental role in pain pathophysiology." This is an overstatement as Chen and colleagues examined the role of these channels in the anterior cingulate cortex in CCI-mediated neuropathic pain (Shen, et al., 2015, Molecular Pain).

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The manuscript "Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain" identifies a subset of parvalbumin-expressing GABAergic neurons in the anterior pretectum (APT) that co-express Cav2.3 T-type calcium channels. The firing frequency and burst patterns are potentiated in these neurons following spared nerve injury (SNI) and the development of neuropathic pain. Deletion of the channels in these cells reduced both the development and maintenance of mechanical and cold allodynia. Studies show nice co-expression of the PV and GFP in the Cav2.3.2eGFP-flox KI mouse line.

    Multi-unit recordings from PV-Cre X Ai32 mice show that PV neurons in the APT are fast-spiking and that the mean firing rate and frequency of spikes in bursts are potentiated in SNI animals. The graphs in Fig. 2, panel F show compiled data of 18-20 cells from 6-8 animals depending on the treatment. The statistical design for the in vivo experiments (and actually all of the studies) are not clearly stated with degrees of freedom. It is important to know if recordings from a single animal are considered independent observations, and if so, what the rationale for that is. This information should be included in the Quantification and Statistical Analysis section. In addition, it would be interesting to determine if T-type calcium channel blockers can reverse this behavior in these recordings.

    In vitro electrophysiological studies show that the PV-expressing APT neurons exhibit fast-spiking to depolarization and single-cell RT-PCR shows that Cav3.2 is expressed in APT neurons that also express GABA. These cells show an after-hyperpolarization burst of APs that is reduced by blockers of Cav3.2 channels. There are no statistics displayed on panels C-E in Fig. 3, although they are reported in the text. Again, the test used and degrees of freedom, etc. should also be reported as it allows for evaluation of the experimental design. It is also noted in the Discussion (lines 185-186) that "Our in vitro data indicate that 92% of APT-PV+ neurons are able to discharge bursts of action potentials at high frequency underpinned by a large transient depolarization due to the activation of T channels." It would be more clear to refer to the rebound as the figure also shows the fast-spiking properties due to depolarization as well as the transient depolarization due to the rebound but only an effect of the Cav2.3 on the rebound.

    Behavioral studies of mechanical and cold allodynia in male and female naïve and SNI-treated KI and KO mice were performed. These results show a clear contribution of the Cav3.2 channels in APT in both the development and maintenance of neuropathic pain. Again, the statistical design is not clearly defined and it is extremely difficult to resolve what comparisons are delineated in panels B-E of Fig. 4.

  5. eLife

    Reviewer #2 (Public Review):

    Using the Cav3.2-eGFP-flox knock-in mice, the authors showed that the expression of Cav3.2 in APT overlaps well with PV. Most of them are bursting neurons with enhanced activities after spared nerve injury (SNI). The authors provided evidence that Ni-sensitive Cav3.2 current contributes to the bursting activity of the PV+ neurons in APT. Deletion of Cav3.2 in APT attenuated mechanical and cold allodynia in SNI mice. These results support the role of supra-spinal Cav3.2 in neuropathic pain. Understanding the role of Cav3.2 in the bursting of APT neurons and in neuropathic pain is of general interest. The experimental approaches are elegant, but some proper controls are missing. Whether Cav3.2 underlies SNI-induced enhancement of bursting is not directly tested. The relative contributions of APT and spinal/peripheral Cav3.2 to neuropathic pain remains unclear.

  6. eLife

    Reviewer #3 (Public Review):

    The authors used state-of-the-art techniques to investigate the role of centrally located (GABAergic APT neurons) CaV3.2 isoform of T-channels in an animal model of neuropathic pain using speared nerve injury model. This is generally an excellent and very rigorous study. The data is very compelling and it is likely going to have a major impact in the field of ion channels and pain transmission. The data presentation is superb and major conclusions are highly justified. Major strengths include the use of powerful complementary techniques such as molecular (single-cell PCR), mouse genetics, and pain testing in vivo, as well as sophisticated ex vivo (slice physiology) and in vivo recordings (burst analysis using tetrodes). This study may explain recent clinical studies that failed to show the efficacy of peripherally acting Cav3.2 channel blockers in patients with neuropathic pain. Hence, this study has the potential to change the focus from peripheral to supraspinal Cav3.2 channels in various pain pathologies.

    Some moderate weaknesses are identified and should be addressed:

    1. The data showing the effect of T-channel deletion on the excitability of GABAergic neurons of APT is very convincing. However, what is missing is a discussion of how changes in the excitability of inhibitory APT neurons impact the circuitry that is involved. Without knowing the circuitry involved, one could speculate that blocking inhibitory drive may do just the opposite effect of what is proposed and increase hyperalgesia.
    2. Methods should clearly state if any experiments were done in a blinded fashion.
    3. There is no mention anywhere of how was selective Cav3.2 knock-out achieved, nor how was this assessed. It would be very helpful if authors could perform recordings of T-channel amplitudes in sham animals, animals after SNI and after selective knock-out in the SNI group.
    4. It should be discussed that global Cav3.2 animals had only minimal neuropathic pain phenotype (Choi et al., 2007).
  7. Version published to 10.1101/2022.04.27.489708v1 on bioRxiv