Involvement of Cav3.2 channels in the development of inflammation and related mechanical hypersensitivity: genetic evidence
Taking into consideration that no pharmacological tool is exclusively specific to Cav3.2 channels, we investigated the contribution of these channels in inflammation and related allodynia/hyperalgesia using mice constitutively deleted of the CACNA1H gene (Cav3.2 KO). In the carrageenan model, Cav3.2 KO mice did not develop allodynia (Figure 2A left ) nor hyperalgesia (Figure 2B left ) compared to their control wild-type (WT) littermates. Edema development was observed in both WT and Cav3.2 KO mice but to a significantly lesser extent in the latter (Figure 2C left ). In addition, the pro-inflammatory cytokine IL-6 level in the edema was significantly lower in Cav3.2 KO mice (Figure 2D left ). In the CFA model, similar results were obtained: a lack of mechanical nociceptive behavior in Cav3.2 KO mice (Figures 2A right and 2B right ) and a significant decrease in both edema size (Figure 2C right ) and IL-6 level in the edema (Figure 2D right ) compared to those in WT animals. Thus, these findings underscore the importance of Cav3.2 subtype channels in inflammation and related allodynia/hyperalgesia.
Functional location of Cav3.2 channels involved in inflammatory pain-like symptoms
Next, we gained further insights into the role of Cav3.2 channels in inflammatory pain by investigating the impact of their inhibition at different locations. We assessed the effect of the deletion of Cav3.2 channels located in a population of primary sensory neurons in mice, named C-LTMRs. These fibers express both the sodium channel Nav1.8 and the calcium channel Cav3.2 (François et al., 2015) and have been shown to contribute to inflammatory pathological pain (Seal et al., 2009; Delfini et al., 2013; François et al., 2015; Reynders et al., 2015; Urien et al., 2017; Bohic et al., 2020). We therefore used a genetic mouse model in which the expression of Cav3.2 channels is conditionally knocked out in C-LTMRs, by crossing Cav3.2GFP-flox KI with Nav1.8cre KI mice as previously described (François et al., 2015). In this mouse model (Cav3.2Nav1.8 cKO), a significant decrease in allodynia and hyperalgesia in the von Frey test was observed after carrageenan (Figure 3A ) or CFA (Figures 3B ) injections compared to control Cav3.2GFP-flox KI littermates. These results evidenced that the Cav3.2 channels located on C-LTMRs are required for the development of inflammatory allodynia/hyperalgesia. This involvement of C-LTMRs-located Cav3.2 channels could arise from their projection in the spinal cord (pre-synaptic Cav3.2 channels) or their origin in peripheral tissue (Cav3.2 channels on primary afferent fibers). Therefore, the contribution of both locations was studied. First, peripheral Cav3.2 channels involvement was evaluated using an intraperitoneal injection of ABT-639 (10, 30 and 100 mg/kg), a peripherally restricted T-type calcium channels inhibitor. After such treatment, we observed a robust analgesic effect on allodynia and hyperalgesia at the two highest doses tested (30 and 100 mg/kg) in both carrageenan (Figure 4A ) and CFA (Figure 4B ) models. This result was confirmed by performing an intraplantar injection of ABT-639 (2.5 µg / mouse in 20 µl) in the CFA model (Figure 4C ). Second, an intrathecal injection of ABT-639 (10 µg / mouse in 5 µl) was performed in the CFA model to determine the contribution of Cav3.2 channels in primary afferent spinal terminals to allodynia/hyperalgesia. The injection partially reduced the responses to the von Frey test (Figure 4D ). Thus, both pharmacological and genetic tools identified the involvement in inflammatory pain of Cav3.2 located in primary afferent neurons at both the peripheral and spinal terminals.