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Comparative Study
. 2012 Feb 8;32(6):2018-26.
doi: 10.1523/JNEUROSCI.5138-11.2012.

Generation of a pain memory in the primary afferent nociceptor triggered by PKCε activation of CPEB

Oliver Bogen  1 , Nicole Alessandri-Haber, Carissa Chu, Robert W Gear, Jon D Levine
Affiliations
Comparative Study

Generation of a pain memory in the primary afferent nociceptor triggered by PKCε activation of CPEB

Oliver Bogen et al. J Neurosci. .

Abstract

Isolectin B(4)-positive [IB(4)(+)] primary afferent nociceptors challenged with an inflammatory or neuropathic insult develop a PKCε-dependent long-lasting hyperalgesic response to a subsequent challenge by the proinflammatory cytokine prostaglandin E(2) (PGE(2)), a phenomenon known as hyperalgesic priming. Here we demonstrate that the neuroplasticity underlying nociceptor priming requires 72 h to be established; rats that have been challenged with the inflammatory mediator TNFα 24 or 48 h ahead of PGE(2) do not show the enhanced and prolonged hyperalgesic response by which primed IB(4)(+)-nociceptors are being characterized. Moreover, as the underlying plasticity can be interrupted by the peripheral administration of the protein translation inhibitor anisomycin it is reflected by changes in the peripheral protein expression pattern. Finally, the induction of priming by the selective PKCε agonist, psi ε receptor for activated c kinase (ψεRACK) can be prevented, but not reversed by intrathecal injections of antisense oligodeoxynucleotides for the cytoplasmic polyadenylation element binding protein (CPEB) mRNA, a master regulator of protein translation that coimmunoprecipitated with PKCε and is almost exclusively expressed by IB(4)(+)-nociceptors. Our results suggest that CPEB is downstream of PKCε in the cellular signaling cascade responsible for the induction of priming, raising the intriguing possiblity that prion-like misfolding could be a responsible mechanism for the chronification of pain.

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Figures

Figure 1.
Figure 1.
Time to onset of priming. TNFα (100 ng) or vehicle was administered intradermally into the hindpaws of separate groups of rats. As previously demonstrated, TNFα-induced hyperalgesia returned to baseline within 24 h following administration (Parada et al., 2003a). To test for the existence of priming, PGE2 (100 ng) was administered into the same site 1, 2, or 3 d following TNFα administration (A–C, respectively). PGE2-induced hyperalgesia returned to baseline within 4 h on days 1 and 2 following TNFα administration and on all 3 d following vehicle administration. However, PGE2-induced hyperalgesia remained elevated at 4 h on day 3 in the TNFα-treated rats, indicating the onset of hyperalgesia priming (Aley et al., 2000; Parada et al., 2003b). A, Day 1. The two-way repeated-measures ANOVA showed no significant differences between the TNFα- and the vehicle-treated groups, either with respect to the group × time interaction (F(1,10) = 0.033, p = 0.861) or main effect of group (F(1,10) = 0.575, p = 0.466). B, Day 2. The two-way repeated-measures ANOVA showed no significant differences between the TNFα- and the vehicle-treated groups, either with respect to the group × time interaction (F(1,10) = 0.007, p = 0.985) or main effect of group (F(1,10) = 0.236, p = 0.638). C, Day 3. The two-way repeated-measures ANOVA showed significant differences between the TNFα- and the vehicle-treated groups, the group × time interaction was F(1,10) = 55.487, p < 0.001, and the main effect of group was F(1,10) = 14.828, p = 0.003, indicating that the onset of hyperalgesic priming is not immediate but occurs after a 3 d delay after TNFα administration.
Figure 2.
Figure 2.
Carrageenan-induced priming can be prevented by anisomycin. Anisomycin (1 μg) or its vehicle was injected intradermally once each day in separate groups. A single dose of carrageenan (5 μl of a 1% solution) was administered to all rats on the first day of anisomycin administration. Carrageenan-induced hyperalgesia returned to baseline by day 4 in both anisomycin- and vehicle-treated rats (data not shown). To test for hyperalgesic priming, PGE2 (100 ng) was administered into the same sites and tested at 30 min, 4 h and 24 h. PGE2-induced hyperalgesia remained elevated to the 24 h time point in vehicle-treated rats, indicating the presence of hyperalgesic priming, but hyperalgesia was dissipated by 4 h in anisomycin-treated rats, demonstrating its ability to block of hyperalgesic priming. The two-way repeated-measures ANOVA showed a significant time × group interaction (F(3,30) = 61.731, p < 0.001) as well as a significant main effect of group (F(1,10) = 193.272, p < 0.001).
Figure 3.
Figure 3.
A, Colocalization of anti-CPEB immunoreactivity and IB4-reactivity in rat L4 DRGs. Anti-CPEB immunoreactivity (left) can be detected in a large subpopulation of small to medium sized sensory neurons. As the majority of cells that express CPEB immunoreactivity can also be labeled with IB4 (middle and right), a marker for a subset of nociceptive C-fiber afferents, many of them are supposed to be nociceptors that innervate the upper epidermal layer of the skin, at the side of nociceptive testing. Red fluorescence, Goat anti-CPEB antibody/Cy3-conjugated anti-goat antibody. Green fluorescence, IB4-FITC conjugate. Blue, DAPI counterstain. Scale bar, 100 μm. B, Antisense ODNs for CPEB mRNA can prevent carrageenan-induced priming. Naive rats were treated with antisense or mismatch ODN for CPEB mRNA (40 μg/day) for 7 consecutive days. A single dose of carrageenan (5 μl of a 1% solution) was administered to all rats on day 4 of ODN administration. On day 8, 1 d after the last ODN administration, carrageenan-induced hyperalgesia had diminished to baseline levels, and PGE2 was administered into the same site, as was carrageenan before. To test for hyperalgesic priming, paw-withdrawal thresholds were measured 30 min, 4 h and 24 h after PGE2 administration. PGE2-induced hyperalgesia remained elevated to the 24 h time point in mismatch-treated rats, indicating the presence of hyperalgesic priming, but hyperalgesia was dissipated by 4 h in antisense-treated rats, demonstrating its ability to block of hyperalgesic priming. A two-way repeated-measures ANOVA showed a significant main effect of time (F(2,20) = 486.280; p < 0.001), indicating that, overall, nociceptive thresholds changed over the times tested, but no significant group × time interaction (F(2,20) = 1.199; p = 0.322) and no significant main effect of group (F(1,10) = 3.341; p = 0.098). These results indicate that the ODN groups did not differ significantly from each other up until the time of PGE2 injection. A second two-way repeated-measures ANOVA that included the three post-PGE2 time points showed a significant group × time interaction (F(5,50) = 68.856; p < 0.001), a significant main effect of group (F(1,10) = 120.561; p < 0.001), as well as a significant main effect of time (F(5,50) = 250.686; p < 0.001). Together these results indicate that the groups differed significantly only after PGE2 administration. C, Downregulation of the peripheral CPEB expression level by antisense ODN for CPEB mRNA. Protein extracts derived from the saphenous nerve of animals that have been treated with intrathecal antisense ODN for CPEB mRNA for 3 consecutive days demonstrate a 28 ± 8% decrease in the anti-CPEB immunoreactivity compared with those that were treated with mismatch ODN (p < 0.05; unpaired Student's t test; N = 6 each for antisense- and mismatch-treated rats). Note that the two bands visualized by anti-CPEB immunoreactivity are most likely due to a phosphorylated (upper band) and nonphosphorylated (lower band) variant. The calculated molecular weight of CPEB is 62 kDa (according to UniProtKB/Swiss-Prot database-entry P0C279).
Figure 4.
Figure 4.
A, PKCε can be coimmunoprecipitated with CPEB (and vice versa). Protein extracts derived from DRGs of carrageenan-primed rats were first immunoprecipitated with an anti-CPEB or anti-PKCε antibody and the precipitates subsequently analyzed by Western blotting using the respective anti-PKCε or anti-CPEB antibodies as described in Materials and Methods. Note that the calculated molecular weight of PKCε is 84 kDa (according to UniProtKB/Swiss-Prot database-entry P09216). B, ψεRACK-induced priming can be prevented by antisense ODN for CPEB mRNA. Naive rats were treated with antisense or mismatch ODN for CPEB mRNA (40 μg/day) for 7 consecutive days. A single dose of ψεRACK (1 μg) was administered to all rats on day 4 of ODN administration. On day 8, 1 d after the last ODN administration PGE2 was administered into the same sites as ψεRACK before. To test for hyperalgesic priming, paw-withdrawal thresholds were measured 30 min, 4 h and 24 h after PGE2 administration. PGE2-induced hyperalgesia remained elevated to the 24 h time point in mismatch-treated rats, indicating the presence of hyperalgesic priming, but hyperalgesia was dissipated by 4 h in antisense-treated rats, demonstrating its ability to block of hyperalgesic priming. A two-way repeated-measures ANOVA showed a significant main effect of time (F(4,40) = 283.485; p < 0.001), indicating that, overall, nociceptive thresholds changed over the times tested, but no significant group × time interaction (F(4,40) = 0.389; p = 0.815) and no significant main effect of group (F(1,10) = 0.019; p = 0.892). These results indicate that the ODN groups did not differ significantly from each other up until the time of PGE2 injection. A second two-way repeated-measures ANOVA that included the three post-PGE2 time points showed a significant group × time interaction (F(7,70) = 66.212; p < 0.001), a significant main effect of group (F(1,10) = 70.272; p < 0.001), as well as a significant main effect of time (F(7,70) = 241.187; p < 0.001). Together these results indicate that the groups differed significantly only after PGE2 administration. C, ψεRACK-induced priming cannot be reversed by antisense ODN for CPEB mRNA. Naive rats received a single dose of ψεRACK (1 μg) on day 1 of the experiment. Beginning on day 5, rats were treated with antisense or mismatch ODN for CPEB mRNA (40 μg/day) for 3 consecutive days. On day 8, 1 d after the last ODN administration, PGE2 was administered into the same site as ψεRACK before. To test for hyperalgesic priming, paw-withdrawal thresholds were measured 30 min, 4 h and 24 h after PGE2 administration. PGE2-induced hyperalgesia remained elevated to the 24 h time point in mismatch- and antisense-treated rats, indicating the presence of hyperalgesic priming in both groups. Thus, antisense for CPEB mRNA was not able to reverse ψεRACK-induced priming. The two-way ANOVA showed that neither the time × group interaction nor the main effect of group were significant (F(3,30) = 0.50; p < 0.985 and F(1,10) = 0.066; p < 0.803, respectively), indicating that the decrease in the CPEB expression level fails to reverse hyperalgesic priming induced by ψεRACK.
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