Fig 2: Effects of ?-conotoxin on peripheral neuronal growth and Cav2.2 protein expression. (A) Representative micrographs show TUJ1 immunolabeling in DRGs. A significant increase in TUJ1 labeled neurites can be seen post CFA inflammation (white arrows), and this is not observed post ?-conotoxin treatment. (B) Representative micrographs show an increase in Cav2.2 protein expression post CFA as revealed by antisera detected against Cav2.2 CFA. This increase in Cav2.2 expression is not observed when combining CFA with ?-conotoxin treatment. N = 5 images per group obtained from three mice. Scale bar = 30 µM. (C,D) Immunoblots show an increase in TUJ1 protein expression in DRGs and paws. (E) Immunoblots show an increase in Cav2.2 protein expression post CFA treatment in DRGs, which is not observed combining CFA with ?-conotoxin treatment. (F) Quantification of TUJ1 and Cav2.2 immunoblots, n = 3 mice per group, *P < 0.05 using one-way ANOVA followed by Holm–Sidak testing for multiple comparisons.

Fig 3: Cav2.2 RNA and protein are upregulated in thermal and not mechanical sensitive nociceptors after CFA-induced inflammation. In situ hybridization with gene specific RNAscope probes of contralateral (non-treated) (A,C,E) and ipsilateral (treated) (B,D,F) DRGs showing Cav2.2 mRNA (green) in heat-sensing TRPV1 + nociceptors (red), cold-sensing TRPM8 + nociceptors (blue), noxious-stimuli sensing TRPA1 + nociceptors (red), and mechanical-sensing MrgprD + nociceptors (red) observed 48 h post CFA induced inflammation. (G–J) Mean relative fluorescence intensity of Cav2.2 mRNA (expressed in “arbitrary units” defined by NIH/ImageJ software) in TRPV1+, TRPA1+, TRPM8+, and MrgprD + nociceptors in ipsilateral and contralateral DRGs. A significant increase in Cav2.2 mRNA in neuronal populations co-expressing TRPV1, TRPA1, and TRPM8, but not MrgprD mRNA, was observed after inflammation. N = 5 sections per group obtained from three mice. *P < 0.05 comparing ipsilateral to contralateral DRGs with student’s t-test. Error bars represent standard error of the mean. (K,L) An increase in total Cav2.2 protein expression was observed in ipsilateral lumbar DRGs after CFA induced inflammation. *P < 0.05 comparing ipsilateral to contralateral DRGs with student’s t-test. Error bars represent standard error of the mean.

Fig 4: Inflammation associated neurite growth is dependent on Cav2.2 activity and is driven via ERK activation. (A) Representative micrographs show sciatic nerve sections from control, CFA treated and CFA + ?-conotoxin treated mice. A significant increase in TUJ1 expression can be seen post CFA treatment (white arrows in inset pointing at TUJ1 labeled afferents). ?-conotoxin prevents CFA mediated increase in TUJ1 labeled afferents. n = 3 mice per group. Scale bar = 30 µM, inset scale bar = 40 µM. (B) Representative micrographs show phosphorylated-ERK1/2 immunolabeling. Conotoxin prevents CFA induced increase in phosphorylated ERK. (C) Immunoblots showing changes in TUJ1and pERK1/2 expression in sciatic nerve consistent with histochemical analysis. Conotoxin mitigates TUJ1 and p-ERK1/2 activity upon CFA induced inflammation, n = 3 mice per group, *P < 0.01 using one-way ANOVA followed by Holm–Sidak testing for multiple comparisons between groups.

Fig 5: Graphical abstract summarizing the main findings of this work which highlight a peripheral role for Cav2.2 during inflammation. CFA induced inflammation leads to increased Cav2.2 expression and ion channel activity which seem to drive peripheral afferent growth at the site of inflammation. One way that these changes could be manifesting is through activation of the ERK1/2-MAPK pathway further downstream. Together, these changes contribute to inflammation associated hyperalgesia. Intrathecal injection of ?-conotoxin alleviates hyperalgesia by blocking Cav2.2 activity and associated afferent growth by downregulating ERK1/2. Schematic representation created with BioRender.com.