Mice lacking central serotonergic neurons show enhanced inflammatory pain and an impaired analgesic response to antidepressant drugs

Abstract

A large body of literature has implicated serotonin [5-hydroxytryptamine (5-HT)] in descending modulation of nociceptive transmission. Here, we have studied the pain behavior of Lmx1b conditional knock-out mice (Lmx1b(f/f/p)), which lack 5-HT neurons in the CNS. Lmx1b(f/f/p) mutant mice showed normal thermal and visceral pain responses but were less sensitive to mechanical stimuli and exhibited enhanced inflammatory pain compared with their littermate control mice. Importantly, the analgesic effect of several antidepressant drugs, including selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and tricyclic antidepressants, was either abolished or greatly attenuated in Lmx1b(f/f/p) mice. Moreover, in the acute versus persistent pain settings, the analgesic actions of the SNRI duloxetine and the SSRI fluoxetine were differentially affected. Together, our results provide in vivo genetic evidence demonstrating that although the predominant role of the central 5-HT system in inflammatory pain is inhibitory, its role in acute mechanical pain is facilitatory. The findings that the analgesic effects of various antidepressant drugs are differentially dependent on the central 5-HT system should help us to understand the mechanism of the analgesic action of different classes of antidepressants in the management of persistent pain.

Figure 1.

Acute pain and visceral pain tests. A, Responses to noxious thermal stimulation, as measured by the tail-flick latency (left) and the paw withdraw latency (Hargreaves, right) induced by radiant heat did not differ between wild-type and Lmx1b^f/f/p mice. B, Left, Paw-withdrawal threshold after exposure to mechanical (von Frey filaments) stimulus was increased in Lmx1b^f/f/p mice compared with wild-type mice (***p < 0.001, 2-tailed t test). Right, Visceral pain in response to intraperitoneal injection of 0.6% acetic acid solution; values represent the number of abdominal stretches (writhes) in 20 min after injection. In all tests, n = 9–15 per genotype. WT, Wild type.

Figure 2.

Enhanced pain behavior in Lmx1b^f/f/p mice after capsaicin injection. A, Left, A significant increase in duration of licking and flinching responses within 5 min after an intraplantar injection of capsaicin in Lmx1b^f/f/p mice (n = 15) compared with wild-type control (n = 14; *p < 0.05, 2-tailed t test). Right, Capsaicin-evoked paw edema measured by the changes of paw diameter 1 h after capsaicin injection (the value 1 h after injection minus the value of the same hindpaw before injection) in Lmx1b^f/f/p mice (n = 9) did not differ from wild-type mice (n = 10). B, Increased mechanical sensitivity measured with von Frey filaments in the ipsilateral hindpaw induced by capsaicin in Lmx1b^f/f/p mice compared with wild-type mice. ^#p < 0.05, two-tailed t test; *p < 0.05, repeated two-way ANOVA. Paw-withdrawal mechanical threshold (in grams) of wild-type mice and Lmx1b^f/f/p mice before or from 30 to 180 min after capsaicin injection was measured, respectively (n = 9–12). ipsi, Ipsilateral; PWT, paw-withdrawal threshold; WT, wild type.

Figure 3.

Enhanced pain behavior in Lmx1b^f/f/p mice after formalin injection. A, The first phase (0–10 min) of pain behavior (as duration of licking and flinching responses) did not differ between wild-type (n = 17) and Lmx1b^f/f/p mice (n = 11) after formalin injection, but the second phase (10–50 min) was significantly enhanced in Lmx1b^f/f/p mice. Data were plotted in 5 min intervals; *p < 0.05, repeated two-way ANOVA, Lmx1b^f/f/p versus wild type. B, Left, Data from A grouped into two phases; *p < 0.05; two-tailed t test; Lmx1b^f/f/p versus wild type. Right, Formalin-evoked paw edema in Lmx1b^f/f/p mice (n = 11) and wild-type mice (n = 17) did not differ. WT, Wild type.

Figure 4.

Inhibitory effects of intrathecal injection of different dosages of 5-HT on wild-type and Lmx1b^f/f/p mice in a capsaicin model of pain. Intrathecal injection of 50 and 500 ng of 5-HT on wild-type mice reduced the capsaicin-evoked licking and flinching behavior but not significantly compared with the vehicle control. In Lmx1b^f/f/p mice, the capsaicin-induced pain behavior was significantly attenuated by intrathecal injection of 5-HT (500 ng) 10 min before capsaicin injection. *p < 0.05, Lmx1b^f/f/p versus wild type; **p < 0.01, 5-HT versus vehicle, one-way ANOVA followed by Tukey's post hoc test. Lmx1b^f/f/p 500 ng group, n = 13; all other groups, n = 7 animals. WT, Wild type.

Figure 5.

5-HT receptor expression in the lumbar spinal cord of Lmx1b^f/f/p mice and wild-type mice. No major difference of 5-HT receptor expression, including 5-HT_1A (A, B), 5-HT_1B (C, D), 5-HT_2A (E, F), 5-HT_2C (G, H), 5-HT_1D (I, J), and 5-HT_3A (K), was found between wild-type (left) and Lmx1b^f/f/p (right) mice. A–H, In situ hybridization data. I, J, Immunocytochemical staining. K, Semiquantitative RT-PCR result in the lumbar spinal cord. Scale bar, 100 μm. WT, Wild type.

Figure 6.

Lack of analgesic efficacy of antidepressants in Lmx1b^f/f/p mice in the acute thermal pain model. A, Fluoxetine injection (20 mg/kg, i.p.) had no effect in Lmx1b^f/f/p mice (n = 9) in the thermal threshold using a Hargreaves-type device, in comparison with that it elicited a strong analgesic effect in wild-type mice (n = 8) that lasted >3 h after injection. B, Amitriptyline injection (20 mg/kg, i.p.) elicited a strong analgesic effect in wild-type mice (n = 8) that lasted for 2 h after injection. The analgesic effect of amitriptyline is strongly attenuated in Lmx1b^f/f/p mice (n = 9). C, Duloxetine injection (30 mg/kg, i.p.) elicited an analgesic effect in wild-type mice (n = 8) that peaked at 60 min after the injection. However, thermal threshold in Lmx1b^f/f/p mice (n = 7) is not affected by duloxetine injection. For each animal, the percentage maximum possible effect (%MPE) was calculated using the following formula: [(postdrug latency − predrug latency)/(cutoff time − predrug latency)] × 100. *p < 0.05; **p < 0. 01, two-way ANOVA followed by Fisher's post hoc test. PWL, Paw-withdrawal latency. D, There were no obvious differences in the levels of NET protein tested by Western blot analyses in the spinal cord of Lmx1b^f/f/p compared with wild-type mice (n = 4). WT, Wild type.

Figure 7.

Antidepressants differentially attenuated the second phase of formalin-induced nociceptive behavior in wild-type mice and Lmx1b^f/f/p mice. A, B, Both fluoxetine (20 mg/kg, i.p.) and duloxetine (30 mg/kg, i.p.) injection significantly inhibited the second phase of pain behavior compared with the vehicle injection in wild-type mice (n = 8–12). C, D, Fluoxetine (20 mg/kg, i.p.) has no significant inhibitory effect on the second phase of formalin pain behavior, but duloxetine (30 mg/kg, i.p.) still has the effect compared with the vehicle injection in Lmx1b^f/f/p mice (n = 8–12). *p < 0.05, two-way ANOVA followed by Fisher's post hoc test. WT, Wild type.

Figure 8.

Antidepressants differentially attenuated carrageenan-induced mechanical hypersensitivity in wild-type mice and Lmx1b^f/f/p mice. A, B, Both wild-type mice (n = 8) and Lmx1b^f/f/p mice (n = 6) developed significantly increased mechanical hypersensitivity as measured by withdrawal threshold in grams to von Frey stimulus, 3 h after intraplantar injection of 2% carrageenan [intraplantar injection of vehicle (saline) as the control for carrageenan and intraperitoneal injection of vehicle (saline) 1.5 h after carrageenan as the control for the antidepressants]. A, Duloxetine (30 mg/kg, i.p.) injection 1.5 h after carrageenan significantly reversed carrageenan-induced mechanical hypersensitivity in both wild-type mice (n = 12) and mutants (n = 9) when compared with the vehicle control of the same genotype. B, Fluoxetine (20 mg/kg, i.p.) significantly reversed mechanical hypersensitivity in wild-type mice (n = 10) but failed to reverse carrageenan-induced mechanical hypersensitivity in Lmx1b^f/f/p mice (n = 7) compared with vehicle-injected control of the same genotype. *p < 0.05; **p < 0.01; ***p < 0. 001, one-way ANOVA followed by Newman–Keuls post hoc test. PWT, Paw-withdrawal threshold; WT, wild type.

Figure 9.

HPLC analysis of 5-HT and 5-HIAA in the brain of Lmx1b^f/f/p mice after saline vehicle, formalin, and carrageenan injections. There were no significant differences in 5-HT or 5-HIAA concentrations in the brain of Lmx1b^f/f/p mice 30 min after formalin (n = 5) or 3 h after carrageenan (n = 5) injection compared with their respective controls (n = 5). One-way ANOVA followed by Newman–Keuls test for comparing the effect of compound with vehicle. wet wt, Wet tissue weight.