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. 2021 Jan 6;41(1):31–46. doi: 10.1523/JNEUROSCI.2160-20.2020

Figure 7.

Figure 7.

Effect of in vivo treatment of fentanyl-primed rats with reversal agents for Type II priming on PGE2-induced sensitization of small DRG neurons. Rats were primed by the systemic administration of fentanyl (30 µg/kg, s.c.) 8 d before preparing neuronal cultures (4 d before reversal agents, followed by 4 d before culture preparation). Recordings were made in small-diameter DRG neurons from fentanyl-primed animals treated intrathecally with the combination of a Src and MAPK inhibitor (SU6656 + U0126; Type II reversal group; light blue bars) and not treated (primed group; dark blue bars). In A–D, bars show pooled magnitudes of decrease in rheobase after PGE2 application (10 and/or 100 nm), relative to baseline (measured before the first application), measured and analyzed in the same way as described in Figure 6, while symbols show individual values. In A–C, values for primed group were repeated from Figure 5A–C for the purpose of comparison. A, Reduction of rheobase in response to 10 and 100 nm PGE2, analyzed regardless of IB4-binding status. In the primed group the effect of 10 and 100 nm PGE2 was significantly greater than in the Type II reversal group (two-way ANOVA; effect of condition: F(1,70) = 16.6, p = 0.0001; Holm–Sidak's post hoc: t(70) = 3.3, **adjusted p = 0.003 for 10 nm; t(70) = 2.5, *adjusted p = 0.02 for 100 nm). Number of cells in primed group: n = 19 for both 10 and 100 nm; in Type II reversal group: n = 18 for both 10 and 100 nm. B, Reduction of rheobase in response to 10 nm PGE2 in weakly IB4+ (“weak”) and IB4– classes (“neg”) neurons. Attenuation of PGE2-induced sensitization after in vivo administration of the combination of a Src and MAPK inhibitor was statistically significant in both neuronal subpopulations (two-way ANOVA: effect of condition F(1,21) = 15.5, p = 0.0008; Holm–Sidak's post hoc: t(21) = 3.4, **adjusted p = 0.005 for weakly IB4+; t(21) = 2.2, *adjusted p = 0.04 for IB4–). Number of cells (weak/neg) in Type II reversal group: 6/6, in primed group: 7/6. C, Reduction of rheobase in response to 100 nm PGE2 in strongly IB4+ (“strong”) and merged weakly IB4+ and IB4– (“weak and neg”) neurons from primed and reversal groups. Two-way ANOVA revealed statistically significant interaction (F(1,33) = 8.8, p = 0.006), indicating differential effects on different neuronal classes. Indeed, statistically significant attenuation in reversal group compared with the primed group occurred in weakly IB4+ and IB4– but not in strongly IB4+ neurons [Holm–Sidak's post hoc: t(33) = 0.74, adjusted p = 0.46 for strong, not significant (ns); t(33) = 4.1, ***adjusted p = 0.0005 for weak and neg]. Number of cells (strong/weak and neg): 6/13 in primed group, 6/12 in Type II reversal group. D, Reduction of rheobase in response to 100 nm PGE2 in strongly IB4+ (strong), weakly IB4+ (weak), and IB4– (neg) neurons from reversal group. Reduction of rheobase in the strongly IB4+ class were significantly greater than in weakly IB4+ and IB4– neurons [one-way ANOVA: F(2,15) = 11.2, p = 0.0010; Tukey's post hoc: q(15) = 4.8, *adjusted p = 0.011 for strongly IB4+ vs weakly IB4+; q(15) = 6.5, **adjusted p = 0.0010 for strongly IB4+ vs IB4–; q(15) = 1.7, adjusted p = 0.48, not significant (ns), for weakly IB4+ vs IB4–]. Number of cells: 6 strong, 6 weak, 6 neg.