Research ArticlePain

Brain-derived neurotrophic factor stimulation of T-type Ca2+ channels in sensory neurons contributes to increased peripheral pain sensitivity

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Science Signaling  24 Sep 2019:
Vol. 12, Issue 600, eaaw2300
DOI: 10.1126/scisignal.aaw2300
  • Fig. 1 BDNF enhances T-currents in TG neurons.

    (A) Representative current traces (left) and analysis (right) of the effects of NiCl2 (Ni2+, 100 μM) on T-currents. Insets, stimulation protocol. Data are means ± SEM; n = 7 cells from three rats. **P < 0.01, paired t test. (B to D) Time course of T-current changes (B), with representative traces [(C), from points a, b, and c in (B)], and quantified T-current density (D) under control conditions, during exposure to BDNF [50 ng/ml (1.9 nM)], and during washout. Data are means ± SEM; n = 8 cells from three rats. **P < 0.01, paired t test. (E) Dose-response curve of T-currents in TG neurons cultured with BDNF. The curved line represents the best-fit line based on nonlinear regression modeling using the Hill equation. Data are means ± SEM; n indicated above each. (F to H) Effect of BDNF (50 ng/ml) on features of T-type channel activation (F) and steady-state inactivation (G), quantified for respective effects on half-maximum potentials for activation/inactivation (V50) (H). Insets, stimulation protocol. Traces are representative, and graph (H) shows means ± SEM; n = 12 cells from four rats. *P < 0.05, paired t test.

  • Fig. 2 TrkB mediates the BDNF-induced increase in T-currents.

    (A) Western blotting analysis of TrkB abundance in rat TG cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase (loading control). Blots are representative of ≥3 experiments. (B) Double-label immunofluorescent staining of TrkB (red) with isolectin B4 (IB4), calcitonin gene-related peptide (CGRP), or neurofilament 200 (NF200), all green, in rat TG sections. Colocalization quantified from 872 cells from three experiments (right). Scale bar, 35 μm. (C and D) Time course of T-current changes (represented, left; quantified, right) in TG neurons after either (C) application of BDNF (50 ng/ml) alone or pretreated with ANA-12 (10 nM), or (D) application of 7,8-dihydroxyflavone (7,8-DHF, 0.5 μM). a and b indicate the points used for the representative current traces, inset. Data are means ± SEM; n = 8 (C) or 9 (D) cells from three rats. *P < 0.05 and **P < 0.01, paired t test.

  • Fig. 3 The BDNF-induced T-current increase requires PI3K.

    (A and B) Time course of changes in T-current amplitude induced by BDNF (50 ng/ml) in TG neurons pretreated with either LY294002 (20 μM A) or LY303511 (20 μM B). a and b indicate points used for representative current traces, inset. Data (C) are means ± SEM; n = 10 cells from four rats and eight cells from three rats, respectively. **P < 0.01, paired t test. (D) Western blotting assessment of AKT activation in TG cells after BDNF (50 ng/ml) alone and with pretreatment with AKT inhibitor III (AKT III) (10 μM). Blots were representative of at least three experiments. *P < 0.05, unpaired t test. (E) As described in (A) to (C) with AKT inhibitor III (10 μM; n = 9 cells from three rats). (F) Western blotting analysis of AKT abundance in negative control shRNA (NC-shRNA) or AKT-shRNA transfected TG cells. Blots (left) are representative of the means ± SEM (right) from at least three experiments. **P < 0.01, unpaired t test. (G) Representative current traces (left) and analysis (right) of the effect of AKT knockdown on BDNF (50 ng/ml)–induced T-currents. Data are means ± SEM, n = 11 to 15 cells from three to four rats. *P < 0.05, one-way ANOVA.

  • Fig. 4 TrkB-mediated stimulation of T-type channels requires the p38-dependent PKA signaling.

    (A) Time course of T-current changes (left) and analysis (right) indicating the effects of BDNF (50 ng/ml) on T-currents in cells pretreated with calphostin C (50 nM). n = 9 cells from three rats. a and b on the plot indicate which points were used for sample traces, inset. (B) Representative current traces (left) and analysis (right) of PMA (20 μM)–induced T-current response in TG cells pretreated with control or calphostin C (50 nM). n = 10 cells from three rats. **P < 0.01, paired t test. (C) Effect of pretreating TG cells with ANA-12 (10 nM), LY294002 (20 μM), or AKT inhibitor III (10 μM) on the BDNF (50 ng/ml)–induced increase in PKA activity, measured by the fluorescence-based PepTag Non-Radioactive Protein Kinase Assay system, in which the phosphorylation of a PKA substrate was detected. Data are means ± SEM from triplicate replicates from each of three experiments. *P < 0.05 and **P < 0.01, one-way ANOVA. (D) As in (A) but pretreated with PKI 6–22 (1 μM). n = 8 cells from three rats. (E) Effects of pretreatment of TG neurons with PKI 6–22 or KT-5720 (both 1 μM; n = 7 cells from three rats) on the BDNF (50 ng/ml)–induced T-type channel response. **P < 0.01, paired t test. (F and G) Western blot analysis of phosphorylated p38 (p-p38), phosphorylated extracellular signal–regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) in TG cells upon BDNF application (50 ng/ml) alone (F) or with LY294002 (20 μM) or KT-5720 (1 μM) pretreatment (G). Blots are representative of at least three separate experiments. *P < 0.05 and **P < 0.01, unpaired t test. (H and I) Graph of PKA activity (H), representative blots (I; left, n = 3), and analysis (I, right) of PKA activation (p-PKA) in TG cells upon application of BDNF (50 ng/ml) with or without pretreatment with SB203580 (10 μM). *P < 0.05 and **P < 0.01, one-way ANOVA. (J to L) Representative traces (J and K) and analysis (L) of T-current changes induced by BDNF (50 ng/ml) in TG cells pretreated with SB203580 or SB202474 (10 μM; n = 8 and 9 cells, respectively, from four rats). a and b as in (A). All data are means ± SEM. **P < 0.01, paired t test.

  • Fig. 5 BDNF induces TG neuronal hyperexcitability.

    (A and B) Representative current traces (left) and analysis (right) showing the effects of BDNF (50 ng/ml) on voltage-gated Na+ channel (Nav) currents (A) and high voltage–activated (HVA) Ca2+ channel currents (B) in TG neurons. Neurons were held at −60 mV and stepped to 0 mV to elicit the currents. Graphs show means ± SEM; n = 8 (A) or 9 (B) cells from three rats. (C and D) As in (A) and (B) on transient outward A-type K+ currents (IA; C, left) and the sustained delayed rectifier K+ current (IDR; C, right). Data (D) are means ± SEM; n = 12 cells from four rats. *P < 0.05, paired t test. (E and F) Effect of BDNF (50 ng/ml), either alone (E; n = 17 cells from four rats) or after pretreatment with ANA-12 (10 nM, n = 9 cells from three rats), LY294002 (20 μM, n = 8 cells from three rats), SB203580 (10 μM, n = 9 cells from four rats), KT-5720 (1 μM, n = 9 cells from three rats), or SB202474 (10 μM, n = 11 cells from three rats) (F), on the action potential (AP) firing rate (spike frequency) of TG neurons. Insets (E, top) indicate the current injection protocol; resting membrane potential of the neurons was maintained at −70 mV. Graphs are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, paired t test. (G and H) As described for (E) when incubated for 10 min at 37°C (G, n = 15 cells from four rats) or pretreated with NiCl2 (Ni2+, 100 μM; H, n = 12 cells from four rats). Graphs are means ± SEM. **P < 0.01, paired t test. (I and J) AP firing rate in TG cells after Cav3.2 knockdown (J), confirmed by Western blotting of TG tissue homogenates after intra-TG injection (I). Data are means ± SEM; n ≥ 3 experiments (I); n = 12 cells from five rats (J). **P < 0.01 versus NC-siRNA, unpaired t test.

  • Fig. 6 Involvement of peripheral TrkB activation in pain hypersensitivity.

    (A) Mechanical hypersensitivity, determined by the escape threshold (ET), after intra-TG injection of BDNF (5 nmol) or vehicle. *P < 0.05 versus vehicle, one-way ANOVA. (B) Effect of pretreatment with ANA-12 (2 nmol; intra-TG) or TTA-P2 (1 nmol) on BDNF-induced mechanical hypersensitivity. *P < 0.05, one-way ANOVA. (C) Effect of CFA injection (20 μl) or normal saline (NS; 20 μl) on ET. *P < 0.05 versus corresponding NS, one-way ANOVA. BL, baseline. (D) TrkB abundance in CFA-treated or control rats, age- and sex-matched. Blots are representative of at least three experiments. *P < 0.05, unpaired t test. (E) Effect of intra-TG injection of vehicle or ANA-12 (2 nmol, arrow) 2 days after CFA on the ET in rats. *P < 0.05 versus corresponding vehicle, one-way ANOVA. (F) Effect of blockade of T-type channels by intra-TG injection of TTA-P2 (1 nmol) on the ANA-12 (2 nmol)–mediated amelioration of mechanical allodynia in CFA rats. $P < 0.05 versus CFA 2 days; **P < 0.01 versus corresponding vehicle; #P < 0.05 versus vehicle 1 hour; two-way ANOVA. (G) Effect of siRNA-mediated knockdown of Cav3.2 versus control (NC-siRNA) on ANA-12 (2 nmol, intra-TG injection; arrow)–induced alleviation of mechanical allodynia (ET) in CFA rats. +P < 0.05 versus CFA 2 days; *P < 0.05 and **P < 0.01 versus corresponding NC-siRNA; #P < 0.05 versus 0 hours (“siRNA”) NC-siRNA; two-way ANOVA. For (A) to (C) and (E) to (G), n = at least seven rats for each group.

  • Fig. 7 Proposed mechanisms of TrkB signaling on T-type channels.

    BDNF acts through the receptor TrkB and subsequently activates PI3K. Several pathways may stimulate T-type channels, including those of PKA, PKC, and MAPK (p38, ERK, and JNK). Data from this study implicate the involvement of the p38-dependent PKA signaling pathway in the stimulation of T-type channels rather than the AKT and PKC pathway. PI3K-dependent p38 signaling may activate PKA, which then phosphorylates Cav3 channels (mainly Cav3.2) to regulate T-currents.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/600/eaaw2300/DC1

    Fig. S1. Abundance of TrkB in rat TGs.

    Fig. S2. Abundance of p-AKT.

    Fig. S3. Knockdown of AKT in rat TGs.

    Fig. S4. Abundance of p38, ERK, and JNK.

    Fig. S5. Change in the abundance of p-p38 induced by BDNF.

    Fig. S6. Abundance of p-PKA.

    Fig. S7. Knockdown of Cav3.2 in rat TGs.

    Fig. S8. Increased abundance of TrkB induced by CFA.

  • This PDF file includes:

    • Fig. S1. Abundance of TrkB in rat TGs.
    • Fig. S2. Abundance of p-AKT.
    • Fig. S3. Knockdown of AKT in rat TGs.
    • Fig. S4. Abundance of p38, ERK, and JNK.
    • Fig. S5. Change in the abundance of p-p38 induced by BDNF.
    • Fig. S6. Abundance of p-PKA.
    • Fig. S7. Knockdown of Cav3.2 in rat TGs.
    • Fig. S8. Increased abundance of TrkB induced by CFA.

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