Abstract
Voltage-gated calcium channels (VGCCs) are important mediators of pain hypersensitivity during inflammatory states, but their role in sensory nerve growth remains underexplored. Here, we assess the role of the N-type calcium channel Cav2.2 in the complete Freund’s adjuvant (CFA) model of inflammatory pain. We demonstrate with in situ hybridization and immunoblotting, an increase in Cav2.2 expression after hind paw CFA injection in sensory neurons that respond to thermal stimuli, but not in two different mechanosensitive neuronal populations. Further, Cav2.2 upregulation post-CFA correlates with thermal but not mechanical hyperalgesia in behaving mice, and this hypersensitivity is blocked with a specific Cav2.2 inhibitor. Voltage clamp recordings reveal a significant increase in Cav2.2 currents post-CFA, while current clamp analyses demonstrate a significant increase in action potential frequency. Moreover, CFA-induced sensory nerve growth, which involves the extracellular signal-related kinase (ERK1/2) signaling pathway and likely contributes to inflammation-induced hyperalgesia, was blocked with the Cav2.2 inhibitor. Together, this work uncovers a role for Cav2.2 during inflammation, demonstrating that VGCC activity can promote thermal hyperalgesia through both changes in firing rates of sensory neurons as well as promotion of new neurite outgrowth.
Highlights
Inflammation-induced pain hypersensitivity is a manifestation of increased sensory input, altered neurotransmitter release in the spinal cord, heightened excitability of neurons, and phenotypic changes brought about by afferent neurons innervating the site of inflammation (Woolf et al, 1997; Lankford et al, 1998)
Comparing the ipsilateral complete Freund’s adjuvant (CFA)-injected side to the control contralateral side, we observed a statistically significant increase in Cav2.2 expression in TrpV1+, TrpM8+, and TrpA1+ nociceptors (Figures 1G–I)
The RNAscope results are consistent with increases in Cav2.2 protein expression during inflammation, and we further confirmed this by performing western blot analyses with an antibody specific to Cav2.2, and we observed a statistically significant increase in protein expression comparing the control versus CFA injected mice (Figures 1K,L)
Summary
Inflammation-induced pain hypersensitivity is a manifestation of increased sensory input, altered neurotransmitter release in the spinal cord, heightened excitability of neurons, and phenotypic changes brought about by afferent neurons innervating the site of inflammation (Woolf et al, 1997; Lankford et al, 1998). In another study in parasympathetic cultured neurons, it has been shown that blockade of calcium influx through L-type and N-type VGCCs as well as transient receptor potential canonical (TRPC) channels, reduces the growth of neurite processes while release from intracellular stores was not significantly affected (Zamburlin et al, 2013). Xenopus live imaging findings, coupled with studies of cultured Xenopus spinal neurons, demonstrate that an optimal frequency of calcium transients regulates neurite extension (Gu and Spitzer, 1995). It remains unclear whether this same phenomenon drives the afferent sensory growth in inflammation, which leads to pain hypersensitivity at the periphery, and which downstream signaling cascades are involved in sensory afferent outgrowth
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