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, 594 (22), 6607-6626

Cav1.2 and Cav1.3 L-type Calcium Channels Independently Control Short- And Long-Term Sensitization to Pain

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Cav1.2 and Cav1.3 L-type Calcium Channels Independently Control Short- And Long-Term Sensitization to Pain

Houda Radwani et al. J Physiol.

Abstract

Key points: L-type calcium channels in the CNS exist as two subunit forming channels, Cav1.2 and Cav1.3, which are involved in short- and long-term plasticity. We demonstrate that Cav1.3 but not Cav1.2 is essential for wind-up. These results identify Cav1.3 as a key conductance responsible for short-term sensitization in physiological pain transmission. We confirm the role of Cav1.2 in a model of long-term plasticity associated with neuropathic pain. Up-regulation of Cav1.2 and down-regultation of Cav1.3 in neuropathic pain underlies the switch from physiology to pathology. Finally, the results of the present study reveal that therapeutic targeting molecular pathways involved in wind-up may be not relevant in the treatment of neuropathy.

Abstract: Short-term central sensitization to pain temporarily increases the responsiveness of nociceptive pathways after peripheral injury. In dorsal horn neurons (DHNs), short-term sensitization can be monitored through the study of wind-up. Wind-up, a progressive increase in DHNs response following repetitive peripheral stimulations, depends on the post-synaptic L-type calcium channels. In the dorsal horn of the spinal cord, two L-type calcium channels are present, Cav1.2 and Cav1.3, each displaying specific kinetics and spatial distribution. In the present study, we used a mathematical model of DHNs in which we integrated the specific patterns of expression of each Cav subunits. This mathematical approach reveals that Cav1.3 is necessary for the onset of wind-up, whereas Cav1.2 is not and that synaptically triggered wind-up requires NMDA receptor activation. We then switched to a biological preparation in which we knocked down Cav subunits and confirmed the prominent role of Cav1.3 in both naive and spinal nerve ligation model of neuropathy (SNL). Interestingly, although a clear mechanical allodynia dependent on Cav1.2 expression was observed after SNL, the amplitude of wind-up was decreased. These results were confirmed with our model when adapting Cav1.3 conductance to the changes observed after SNL. Finally, our mathematical approach predicts that, although wind-up amplitude is decreased in SNL, plateau potentials are not altered, suggesting that plateau and wind-up are not fully equivalent. Wind-up and long-term hyperexcitability of DHNs are differentially controlled by Cav1.2 and Cav1.3, therefore confirming that short- and long-term sensitization are two different phenomena triggered by distinct mechanisms.

Figures

Figure 1
Figure 1. Computational model of DHN discharge pattern with or without LTCs
A, Cav1.2 (a and c) and Cav1.3 (e and i) are expressed in different subcellular compartments of deep dorsal horn neurons (white stars) as shown with Map2 co‐detection (b, d, g and k). Cav1.2 labelling is restricted to the soma (double arrows in a and c). Cav1.3 is found in the cell body but also in proximal (double arrowheads in e and i) and more distal dendrites [double arrowheads in e and i inset (labelled f and j)]. No changes in localization are observed after nerve injury (SNL, c and i and inset labelled j) compared to control conditions (Sham, a and e and inset labelled f). Scale bar = 10 μm. B1 and B2, DHN discharge pattern without LTCs. B1, DHNs response to application of a square current pulse (2 s). Note that DHN presents a tonic response with a constant discharge frequency. B2, DHN response to a series of eight short (500 ms) square current pulses. Response of DHN was the same for all current pulses. C1 and C2, DHN discharge pattern with LTCs. In response to a square current pulses (2 s), DHN discharge frequency progressively increased showing a plateau potential, followed by PD (C1). Repetitive square current pulses (500 ms × 8) induced a progressive increase in the DHN response followed by PD revealing a wind‐up of DHN discharge (C2). D, normalized response. E, wind‐up coefficient.
Figure 2
Figure 2. Role of Cav1.2 and Cav1.3 on plateau potentials and wind‐up in a computational model of DHNs
A1 and A2, DHN discharge pattern after Cav1.2 removal. With Cav1.3, DHNs exhibit plateau potential (A1) and wind‐up (A2). A3, amplitude of plateau potentials according to the amplitude of the Cav1.3 conductance and the amplitude of the stimulation (amplitude of plateau is measured as the difference in discharge frequency at the end of the stimulus vs. discharge frequency at the beginning of the stimulus). Plateau appears at low stimulus intensity (40 pA) and low conductance (0.5 μS cm–2). A peak of plateau amplitude is reached for a stimulation intensity of 40 pA and a conductance of 10 μS cm–2. A PD appeared at 5 μS cm–2 for a stimulation intensity of 30 pA. B1 and B2, DHN discharge pattern after Cav1.3 removal. Cav1.3 removal converts a plateau DHN into a tonic DHN (B1). No wind‐up is observed (B2). B3, in the absence of Cav1.3 in dendrites, neither plateau potentials, nor wind‐up was observed, regardless the intensity of stimulation. C, normalized response. Wind‐up is still present when Cav 1.3 subunit is present alone but is suppressed when Cav 1.2 is removed. D, wind‐up coefficient. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3. Computational model of DHNs response to repetitive C‐fibre stimulations
A1, DHNs with LTCs (Cav1.2+/Cav1.3+) respond to repetitive C‐fibre stimulations by a progressive increase in the number of spikes followed by PD revealing wind‐up (arrows indicate C‐fibre stimulations). B, Cav1.2 (Cav1.2–/Cav1.3+) removal does not suppress the wind‐up induced by repetitive C‐fibre stimulations. C, Cav1.3 removal (Cav1.2+/Cav1.3–) strongly reduces wind‐up and no PD is observed. D1 and E1, normalized responses plotted against the stimulation number in each condition. D2 and E2, wind‐up coefficient.
Figure 4
Figure 4. Wind‐up also has a synaptic component
A, response of the simulated DHN to repetitive stimulation of C‐fibres when Cav1.2 and Cav1.3 are absent. B, same protocol but NMDA receptors are also removed. C1, normalized response showing that NMDA receptors mediate a slight wind‐up (filled circle) that is completely suppressed when NMDA receptors are suppressed (open circles). C2, wind‐up coefficient.
Figure 5
Figure 5. The onset of wind‐up depends on LTCs in both young and adult rats
AD, EMG recordings of the biceps femoris response to electrical stimulation of the ipsilateral paw in the region of the sural nerve. A and C, in adult rats, before and after intrathecal injection of nicardipine. B and D, in young rats, before and after intrathecal injection of nicardipine. E1, normalized responses plotted against the stimulation number in young and adult rats. E2, wind‐up coefficient in adult and young rats. F1, normalized responses plotted against the stimulation number in adult rats before and after injection of Nicardipine. F2, wind‐up coefficient. G1, normalized responses plotted against the stimulation number in young rats before and after injection of nicardipine. G2, wind‐up coefficient.
Figure 6
Figure 6. In vivo knockdown of Cav subunits
A, quantitative RT PCR of Cav1.2 and Cav1.3 expression following injection of anti‐sense PNA molecules. PNA injection elicits a specific blockade upon the anti‐sense injected. BD, Cav1.2 and Cav1.3 immunostaining after intrathecal injection of anti‐sense molecules targeting Cav1.2 or Cav1.3 mRNA. Note that Cav1.2 labelling is largely decreased after injection of anti‐Cav1.2 PNA, whereas Cav1.3 labelling is not affected (B1, B2 and C). Reciprocally, Cav1.3 labelling is decreased after injection of AntiCav1.3 PNA, whereas Cav1.2 remained unmodified (B3, B4 and D). In (B), arrows indicate cell bodies in the dorsal horn of the spinal cord.
Figure 7
Figure 7. Cav1.3 is responsible for the onset of the wind‐up of a nociceptive flexion reflex
A1A4, EMG recordings of the nociceptive flexion reflex in shams, mismatch‐, anti‐Cav1.2‐ or anti‐Cav1.3‐injected rats (55–60 g). B1, normalized responses plotted against the stimulation number in shams and mismatch. B2, wind‐up coefficient. C1, normalized responses plotted against the stimulation number in shams and Anti‐Cav1.2 PNA injection. C2, wind‐up coefficient. D1, normalized responses plotted against the stimulation number in shams and Anti‐Cav1.3 PNA. D2, wind‐up coefficient.
Figure 8
Figure 8. Decrease in Cav1.3 expression and wind‐up amplitude in SNL rats
A1, normalized responses of WDR neurons plotted against the stimulation number in naive (black dots, sham (grey dots) and SNL rats (white dots). A2, wind‐up coefficient. B, quantitative RT‐PCR reveals a two‐fold increase of Cav1.2 expression and a decrease by ∼40% of Cav1.3 expression. C1, regulation of Cav1.2 (a and b) and Cav1.3 (c and d) expression in deep dorsal horn neurons (white stars). C2, Cav 1.2 staining is regulated in more intense in neuropathic conditions (SNL, double arrows in C1b) than in control rats (Sham, double arrows in C1a). C3, conversely, Cav1.3 labelling is weaker after nerve injury (SNL, double arrows in C1d) than in control rats (Sham, double arrows in C1c). Scale bar = 10 μm. D1, normalized responses of modelized DHN plotted against the stimulation number in naive LTCs proportion (black dots) and in SNL LTCs proportions (white dots). D2, wind‐up coefficient.
Figure 9
Figure 9. SNL‐induced mechanical allodynia is supressed following Anti‐Cav1.2 injection
A, SNL procedure (see Methods) induced a significant decrease in the mechanical threshold response 7 days after the nerve ligation (day 7, P < 0.001 vs. day 0), Repeated‐measures ANOVA followed by Bonferroni's multiple comparison test.). SNL‐induced allodynia is suppressed by repetitive injection of Anti‐Cav1.2 PNA (day 11, P < 0.05 vs. day 0, repeated‐measures ANOVA followed by Bonferroni's multiple comparison test). B, injection of Anti‐Cav1.3 PNA has no effect on SNL‐induced allodynia that persist after repetitive injections at day 11 (day 11, P < 0.001 vs. day 0, repeated‐measures ANOVA followed by Bonferroni's multiple comparison test) C, injection of mismatch PNA does not modify the SNL‐induced allodynia (day 11, P < 0.05 vs. day 0, repeated‐measures ANOVA followed by Bonferroni's multiple comparison test).
Figure 10
Figure 10. In SNL rats, the onset of wind‐up also depends on Cav1.3
A1, normalized responses of WDR neurons plotted against the stimulation number in SNL rats and in SNL rats injected with anti‐Cav1.3 PNA. A2, wind‐up coefficient. B1, normalized responses of WDR neurons plotted against the stimulation number in SNL rats and in SNL rats injected with anti‐Cav1.2 PNA. B2, wind‐up coefficient. C1, normalized responses of WDR neurons plotted against the stimulation number in SNL rats and in SNL rats injected with mismatch PNA. C2, wind‐up coefficient.
Figure 11
Figure 11. The mathematical approach suggest that plateau potentials and wind‐up are not equally affected by changes in LTCs expression
A, a large square current pulse (2 s) elicits a plateau potential in the naive LTC proportion (A1), as well as in case of decreased Cav1.3 and increased Cav1.2 expression (SNL LTC proportion) (A2). Note that neurons were more responsive to the same stimulus intensity but the difference in instantaneous frequency between the beginning of the square pulse and the end of the square pulse remains similar. The amplitude of plateau is not changed. A3, in the naive LTC proportion, as well as in SNL‐LTC conditions, plateaus are dependent on stimulus intensity. The maximal plateau amplitude is reached for a lower stimulus intensity in the SNL LTC proportion than in naive LTC proportions. B1, wind‐up elicited by repetitive square current pulses with the naive LTC proportion. B2, wind‐up elicited by repetitive square current pulses with the SNL LTC proportion. B3, wind‐up coefficient plotted against the pulse intensity. Note that, with higher pulse intensity, wind‐up coefficient is decreased in the SNL LTC proportion.

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