Prolactin Regulates Pain Responses via a Female-Selective Nociceptor-Specific Mechanism

Abstract

Many clinical and preclinical studies report an increased prevalence and severity of chronic pain among females. Here, we identify a sex-hormone-controlled target and mechanism that regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hind paw or spinal cord substantially reduced hyperalgesia in inflammatory models. This effect was mimicked by sensory neuronal ablation of Prlr. Although Prlr mRNA is expressed equally in female and male peptidergic nociceptors and central terminals, Prlr protein was found only in females and PRL-induced excitability was detected only in female DRG neurons. PRL-induced excitability was reproduced in male Prlr⁺ neurons after prolonged treatment with estradiol but was prevented with addition of a translation inhibitor. We propose a novel mechanism for female-selective regulation of pain responses, which is mediated by Prlr signaling in sensory neurons via sex-dependent control of Prlr mRNA translation.

Keywords: Female Reproductive Endocrinology; Sensory Neuroscience.

Graphical abstract

Figure 1

Exogenous PRL-Induced Hypersensitivity in Female and Male Mice (A–D) PRL-induced heat (A and C) and mechanical (B and D) hypersensitivity was assessed at 1 h post-PRL-administration time point in male and estrous female mice. PRL was administrated into the hind paw (ipl; A and B) or intrathecal space of spinal cord (SC; C and D). PRL dosages (0.1, 1, or 10 μg) and sex of mice are indicated. Mechanical threshold was measured with the Dynamic Plantar Aesthesiometer. “Cont” indicates contralateral injection of 1 μg PRL and measurements of hyperalgesia in the ipsilateral hind paw. (E and F) PRL (1 μg) was injected ipl (intra-plantar; panel E) or it (intrathecal; panel F), and mechanical hypersensitivity was measured in males and females at different estrous phases (diestrus [Diestr], estrous [Estr], and proestrus [Proestr]). BL is baseline reading before PRL administration. Data are represented as mean ± SEM. Statistical test is regular two-way ANOVA with Tukey's post hoc test (n = 5–10; NS, non-significant; *p < 0.05; **p < 0.01; #p < 0.0001). See also Figure S1.

Figure 2

Suppression of Postoperative Pain by Prlr Antagonist in Female and Male Mice Vehicle (Veh) or Prlr antagonist (5 μg; ΔPRL) was injected into hind paw (ipl) of male and estrous female mice at 1 day post incision (Inc) or sham procedures. Heat (A) and mechanical (B) hypersensitivity was assessed at 1 h post Veh/ΔPRL injection. Vehicle or ΔPRL (5 μg) was injected intrathecally (it) into spinal cord of male and estrous female mice at 1 day post incision or sham procedures. Heat (C) and mechanical (D) hypersensitivity was assessed at 1 h post Veh/ΔPRL injection. BL are baseline values before incision procedures. Procedures and animal sex are indicated below the x axis. Data are represented as mean ± SEM. Statistical test is regular two-way ANOVA with Tukey's post hoc test (NS, p > 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n = 5–7). See also Figure S2.

Figure 3

Effects of Prlr Antagonist and Agonist in Different Inflammatory Pain Models in Female and Male Rats and Mice (A) Vehicle (Veh) or ΔPRL (5 μg) was injected into spinal cord of male, diestrus female (D-female), or estrous female (E-female) rats at 1 day post incision surgery (POP) or sham procedures. Mechanical hyperalgesia was assessed with Dynamic Plantar Aesthesiometer at 1 h post Veh/ΔPRL injection. BL are baseline values. Statistical test is two-way ANOVA with Tukey's post hoc test (NS, p > 0.05; *p < 0.05; ****p < 0.0001; n = 5–6). (B) IL-6 (1 ng) was injected into hind paw, and vehicle or ΔPRL (5 μg) was injected approximately simultaneously into hind paw (paw) or spinal cord (it) of estrous female mice. Mechanical hyperalgesia was assessed at 1 h post IL-6/Veh or IL-6/ΔPRL co-injections. BL is baseline value. Data are represented as mean ± SEM. Statistical test is regular two-way ANOVA Bonferroni's post hoc test (NS, p > 0.05; ****p < 0.0001; n = 6). (C and D) In the model of hyperalgesic priming, 0.5 ng PRL produces mechanical hypersensitivity in IL-6 (0.1 ng)-primed females (panel C) but not males (panel D). RB is baseline. Statistical test is regular two-way ANOVA Bonferroni's post hoc test ***p < 0.001; ^# p < 0.0001; n = 5). (E) IL-6 (1 ng) and vehicle or PRL (1 μg) were co-injected into the paw in estrous-phase female mice. Mechanical hyperalgesia was assessed at indicated time points. BL is baseline value. Data are represented as mean ± SEM. Statistical test is regular two-way ANOVA Bonferroni's post hoc test (**p < 0.01; ***p < 0.001; ^#p < 0.0001; n = 6).

Figure 4

Hypersensitivity in Inflammatory Pain Models in Sensory Neuronal Prlr CKO Male and Female Mice (A) Postoperative (POP) heat hypersensitivity was measured 1 day post incision in Prlr^fl/fl (lox; control) and Nav1.8^cre/-/Prlr^fl/fl (CKO) female and male mice. (B) POP mechanical hypersensitivity was measured 1 day post incision in lox and CKO female and male mice. (C) IL-6 (1 ng)-induced mechanical hyperalgesia was measured 3 h post IL-6 (ipl) in lox and CKO female and male mice. For (A)–(C), Lox BL and CKO BL are baseline measurements in indicated mouse lines. For (A)–(C), data are represented as mean ± SEM and the statistical test is regular two-way ANOVA with Tukey's post hoc test (NS, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001; n = 5–7). (D and E) Development of IL-6-induced heat (D) and mechanical (E) hyperalgesia in Prlr LOX (control) and Prlr CKO male mice. (F and G) Development of IL-6-induced heat (F) and mechanical (G) hyperalgesia in Prlr LOX and Prlr CKO female mice. BL are baseline measurements in indicated mouse lines. For (D)–(G), data are represented as mean ± SEM and the statistical test is regular two-way ANOVA with Bonferroni's post hoc test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n = 5–6). See also Figures S3 and S4.

Figure 5

Real-time Single Cell Quantitative PCR for Prlr-L and Prlr-S from Prlr-cre⁺ Female and Male DRG Neurons (A) Representative heatmap showing Ct values generated by single-cell RT-PCR from Prlr-cre⁺ female (F) and male (M) DRG neurons. Y axis shows amplified set of genes. X axis marks randomly picked cells for PCR. Values of ≥38 on the heatmap is considered as no amplification. Normalized mRNA expression levels of Prlr-L (B) and Prlr-S (C) isoforms in sensory neuronal groups. Groups for single Prlr-cre⁺ neurons from female and male mouse DRG are indicated on the x axis. Data are represented as mean ± SEM. Statistical test is unpaired t test (non-significant p > 0.05; n = 6–32 depending on Prlr-cre⁺ neuronal group) for each sensory neuronal group.

Figure 6

Sex and Estrogen-dependent Regulation of PRL-induced Excitability of Prlr-cre⁺ DRG Neurons Exogenous PRL induces regulation of excitability in female (A) and male (B) DRG neurons. DRG neurons in culture were treated with vehicle (E2-) or 17β-estradiol (E2+; 1 μg/mL) for 6–36 h in culture. Y axis is change in action potential (AP) frequency (i.e., excitability) after treatment with vehicle or PRL. Data are represented as mean ± SEM. The statistical test is one-way ANOVA with Tukey's post hoc test separately for females or males and for vehicle- or E2-treated groups (*p < 0.05; **p < 0.01; n = 4–12). Examples of AP trains before and after treatment with PRL for 2–3 min are shown in female CGRP⁻/trpV1⁺ E2-treated DRG neurons (C); female CGRP⁻/trpV1⁺ vehicle-treated DRG neurons (D); female CGRP⁺/trpV1⁺ vehicle-treated DRG neurons (E); male CGRP⁻/trpV1⁺ E2-treated DRG neurons (F); and male CGRP⁺/trpV1⁺ E2-treated DRG neurons (G).

Figure 7

E2-controlled Prlr mRNA Transcription and Translation in Male and Female DRG Neurons (A and B) Expression of Prlr-L and Prlr-S mRNA in male (A) and female (B) DRG tissues that was isolated from mice in vivo treated with vehicle (E2^-) or E2 (E2⁺) for 7 days. mRNA levels were assessed by quantitative RT-PCR. Data were analyzed by one-way ANOVA (n = 3–4). (C) Inhibition of PRL (1 μg)-induced mechanical hypersensitivity in female mice by spinal treatment for 1–72 h with translation inhibitor 4EGI-1 (10 μg). BL is baseline read before PRL and 4EGI-1 treatment. “None” is no treatment with 4EGI-1. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (NS, p > 0.05; **p < 0.01; n = 5). (D) PRL (0.2 μg/mL)-induced increase in excitability in female Prlr-cre⁺ cultured DRG neurons pre-treated for 16–20 h with 4EGI-1 (1 μg/mL). Data are represented as mean ± SEM. Statistical test is regular two-way ANOVA with Tukey's post hoc test (variables are treatments with Veh/PRL and Media/4EGI-1; ***p < 0.001; n = 4–8). (E) PRL (0.2 μg/mL)-induced excitability of male Prlr-cre⁺ DRG neurons pre-treated for 16–20 h with mixtures of indicated drugs. Data are represented as mean ± SEM. Statistical test is one-way ANOVA with Tukey's post hoc test (**p < 0.01; n = 6–13). See also Figure S5.

Figure 8

Prlr mRNA Reporter Expression and Prlr Protein Localization in Female and Male Mouse Spinal Cord (A) IHC with Prlr antibodies (polyclonal) and CD68 (rat monoclonal) on spinal cord sections from Prlr^cre/+/Rosa26^{LSL-tDTomato/+} female and male mice. (B) Intensity of TdTomato (Prlr-cre) labeling in spinal cord of female and male Prlr^cre/-/TdTomato mice. (C) Intensity of Prlr protein (Prlr-ab) labeling in spinal cord of female and male Prlr^cre/-/TdTomato mice. Bgr is normalized intensity of background. (D) Intensity of Prlr protein (Prlr-ab) labeling in spinal cord of female and male rats. A representative scale bar of 50 μm is shown. Data are represented as mean ± SEM. Statistical test is unpaired t test (B and D) or one-way ANOVA with Tukey's post hoc test (panel C) (NS, p > 0.05; *p < 0.05; **p < 0.01; n = 3). See also Figure S6.