Dysregulation of serum prolactin links the hypothalamus with female nociceptors to promote migraine

Abstract

Migraine headache results from activation of meningeal nociceptors, however, the hypothalamus is activated many hours before the emergence of pain. How hypothalamic neural mechanisms may influence trigeminal nociceptor function remains unknown. Stress is a common migraine trigger that engages hypothalamic dynorphin/kappa opioid receptor (KOR) signalling and increases circulating prolactin. Prolactin acts at both long and short prolactin receptor isoforms that are expressed in trigeminal afferents. Following downregulation of the prolactin receptor long isoform, prolactin signalling at the prolactin receptor short isoform sensitizes nociceptors selectively in females. We hypothesized that stress may activate the kappa opioid receptor on tuberoinfundibular dopaminergic neurons to increase circulating prolactin leading to female-selective sensitization of trigeminal nociceptors through dysregulation of prolactin receptor isoforms. A mouse two-hit hyperalgesic priming model of migraine was used. Repeated restraint stress promoted vulnerability (i.e. first-hit priming) to a subsequent subthreshold (i.e. second-hit) stimulus from inhalational umbellulone, a TRPA1 agonist. Periorbital cutaneous allodynia served as a surrogate of migraine-like pain. Female and male KORCre; R26lsl-Sun1-GFP mice showed a high percentage of KORCre labelled neurons co-localized in tyrosine hydroxylase-positive cells in the hypothalamic arcuate nucleus. Restraint stress increased circulating prolactin to a greater degree in females. Stress-primed, but not control, mice of both sexes developed periorbital allodynia following inhalational umbellulone. Gi-DREADD activation (i.e. inhibition through Gi-coupled signalling) in KORCre neurons in the arcuate nucleus also increased circulating prolactin and repeated chemogenetic manipulation of these neurons primed mice of both sexes to umbellulone. Clustered regularly interspaced short palindromic repeats-Cas9 deletion of the arcuate nucleus KOR prevented restraint stress-induced prolactin release in female mice and priming from repeated stress episodes in both sexes. Inhibition of circulating prolactin occurred with systemic cabergoline, a dopamine D2 receptor agonist, blocked priming selectively in females. Repeated restraint stress downregulated the prolactin receptor long isoform in the trigeminal ganglia of female mice. Deletion of prolactin receptor in trigeminal ganglia by nasal clustered regularly interspaced short palindromic repeats-Cas9 targeting both prolactin receptor isoforms prevented stress-induced priming in female mice. Stress-induced activation of hypothalamic KOR increases circulating prolactin resulting in trigeminal downregulation of prolactin receptor long and pain responses to a normally innocuous TRPA1 stimulus. These are the first data that provide a mechanistic link between stress-induced hypothalamic activation and the trigeminal nociceptor effectors that produce trigeminal sensitization and migraine-like pain. This sexually dimorphic mechanism may help to explain female prevalence of migraine. KOR antagonists, currently in phase II clinical trials, may be useful as migraine preventives in both sexes, while dopamine agonists and prolactin/ prolactin receptor antibodies may improve therapy for migraine, and other stress-related neurological disorders, in females.

Keywords: hypothalamus; kappa opioid receptors; migraine; prolactin; stress.

Figure 1

Repeated RS-induced CA and LS revealed by UMB inhalation in female and male mice. Data represent tactile responses pooled from all RS and sham female and male mice receiving vehicle treatment or control manipulation from a collection of all performed experiments. Tactile frequency of response was collected at baseline (0) in female (A) and male (C) mice, followed by three consecutive Days of RS for 2 h each. CA was assessed on indicated days after the initial RS over a time course of 16 days (A and C). On Day 16, baseline (0) response was collected, followed by inhalational exposure of UMB (0.01 M/500 µl, each) with measurements collected hourly for 5 h (B female and D male). Data are presented as mean ± SEM and analysed using two-way ANOVA followed by Sidak’s multiple comparison test with *P < 0.05 (n = 49–87). Details of statistical analysis are found in Supplementary Table 1.

Figure 2

KOR^Cre and TH expression in the hypothalamus of female and male mice. (A) Schematic of Cre-dependent expression of Sun1-GFP fusion protein labelling KOR^Cre neurons in KOR^Cre; R26^lsl-Sun1-GFP mice. (B and C) Representative images showing colocalization of KOR^Cre labelled neurons (green) and TH⁺ neurons (red) in hypothalamus from (B) female and (C) male KOR^Cre; R26^lsl-Sun1-GFP mice. Scale bars, 100 μm. (D and E) Higher magnification images showing localization of KOR^Cre (green), TH (red) and DAPI (blue) in the ARC from (D) female and (E) male KOR^Cre; R26^lsl-Sun1-GFP mice. Scale bars, 50 μm. (F and G) Percentages of TH-positive cells colocalized with KOR^Cre neurons in the ARC from (F) female and (G) male mice.

Figure 3

Chemogenetic manipulation of KOR^Cre neurons in the ARC produced increased levels of circulating PRL, transient CA and LS, mimicking effects of RS in female mice. (A) Representative images from KOR^Cre female mice expressing Gi-DREADD (hM4D(Gi)-mCherry) demonstrating expression of hM4D(Gi)-mCherry (red) in the ARC. Scale bars, 100 μm. (B) Concentration of PRL determined from serum collected 30 min after intraperitoneal administration of CNO at 5 mg/kg or vehicle, 4 weeks after stereotaxic administration of AAV8-hSyn-DIO-hM4D(Gi)-mCherry (100 nl) in the ARC from KOR^Cre or KOR^WT female mice. (C and D) Tactile frequency of response at baseline (−1) was collected right before injection of AAV8-hSyn-DIO-hM4D(Gi)-mCherry (100 nl) in the ARC of KOR^Cre or KOR^WT female mice. Four weeks after stereotaxic surgery, tactile frequency of response at baseline (0) was collected followed by three consecutive days of intraperitoneal administration of CNO at 5 mg/kg or vehicle. CA was assessed on indicated days after the first injection over a time course of 16 days (C and D). On Day 16, baseline (0) response was collected, followed by inhalational exposure of UMB (0.01 M/500 µl, each) with measurements collected hourly for 5 h (E, heterozygous and F, wild-type mice). Data are presented as mean ± SEM and analysed using one-way ANOVA followed by Tukey’s (B) or two-way ANOVA followed by Sidak’s (E and F) multiple comparison tests with *P < 0.05 (n = 5–7). Details of statistical analysis are found in Supplementary Table 1.

Figure 4

KORs in the ARC are required for RS-induced periorbital CA and LS revealed by UMB in female mice. (A) Representative western blot of KOR-CRISPR–Cas9 gene editing in ARC of female mice. (B) Quantitative verification of KOR-CRISPR–Cas9 editing efficiency in ARC of female mice. (C) Stereotaxic administration of KOR-CRISPR–Cas9 plasmid (500 nl) in the bilateral ARC blocked RS-induced increased levels of circulating PRL in female mice. Serum was collected 2 h after RS. (D) KOR-CRISPR–Cas9 editing in the ARC blocked RS-induced periorbital CA in female mice. Baseline tactile frequency of response was collected before (−1) and 2 weeks after (0) KOR-CRISPR–Cas9 administration. Following three consecutive episodes of RS, CA was assessed on indicated days over a time course of 16 days. (E) KOR-CRISPR–Cas9 editing in the ARC blocked RS-induced LS revealed by lack of effect of UMB exposure. On Day 16, baseline (0) response was collected, followed by inhalational exposure of UMB with measurements collected hourly for 5 h. Data are presented as mean ± SEM and analysed using a Mann–Whitney U-test (B), one-way ANOVA with Tukey’s (C) or two-way ANOVA followed by Sidak’s (D and E) multiple comparison tests with *P < 0.05 (n = 5–12). Details of statistical analysis are found in Supplementary Table 1.

Figure 5

Increased circulating levels of PRL are required for RS priming-induced periorbital CA and LS only in female mice. (A) RS on three consecutive days resulted in increased levels of circulating PRL in female and male mice, which was prevented by intraperitoneal administration of cabergoline at 1.2 mg/kg, 2 h before each RS episode. Serum was collected immediately after the 2 h of RS. (B and E) Tactile frequency of response was assessed at baseline (0) and on indicated days after the first RS over a time course of 16 days. On Day 16 and again on Day 21 (cabergoline-free), baseline (0) response was collected, followed by inhalational exposure of UMB and CA measurements for 5 h (C, female and F, male). Intraperitoneal treatment with cabergoline or vehicle, were performed 2 h before each RS (on Days 0, 1 and 2) and on Days 4, 6, 8, 10,12, 14, 16 and 17. Cabergoline administration blocked stress priming-induced CA and LS in (B–D) female, but not (E–G) male mice. Data are presented as mean ± SEM and analysed using one- or two-way ANOVA followed by Tukey’s multiple comparison tests *P < 0.05 (n = 5–15). Details of statistical analysis are found in Supplementary Table 1.

Figure 6

PRLRs in the TGV1 are required for stress-induced CA. Repeated RS or chemogenetic manipulation of KOR^Cre neurons in the ARC induced down-regulation of PRLR-long form in trigeminal ganglia V1 region (TGV1) of female mice. (A and C) Representative western blot (WB) images of PRLR isoforms protein expression in TGV1 tissues collected on Day 15 after 3 days of RS episodes (2 h/day) from (A) female and (C) male mice. (B and D) Quantification of PRLR long and short isoform protein expression in TGV1 tissues from (B) female and (D) male mice after RS. (E) Representative WB images of PRLR isoforms protein expression in TGV1 tissues collected on Day 15 after 3 days of CNO injection (5 mg/kg, i.p.), 2 weeks after stereotaxic administration of AAV8-hSyn-DIO-hM4D(Gi)-mCherry (100 nl) in the ARC from KOR^Cre or KOR^WT female mice. (F) Quantification of PRLR long and short isoform protein expression in TGV1 tissues from (E) female KOR^Cre mice after CNO administration. (G) Intranasal administration of PRLR-CRISPR–Cas9 editing in the TGV1 blocked RS-induced periorbital CA in female mice. Tactile frequency of response at baseline (−1) was collected before 2 days of intranasal administration of PRLR-CRISPR–Cas9 or control plasmid (30 µl, each mouse/day) to female mice. Two weeks after the second intranasal administration, tactile frequency of response at baseline (0) was collected followed by three consecutive episodes of RS for 2 h, each. CA was assessed on indicated days after the first injection over a time course of 16 days. (H) PRLR-CRISPR–Cas9 editing in the TGV1 blocked RS-induced LS, revealed by a lack of effect of UMB exposure on restoring periorbital allodynia in female mice. On Day 16, baseline (0) response was collected, followed by inhalational exposure of UMB (0.01 M/500 µl, each) with measurements collected hourly for 5 h. Data are presented as mean ± SEM and analysed using a Mann–Whitney U-test with *P < 0.05 in comparison to control (B–F; n = 4–6) and two-way ANOVA followed by Sidak’s multiple comparison tests with *P < 0.05 (G and H; n = 7–8). Details of statistical analysis are found in Supplementary Table 1.

Figure 7

Anti-CGRP mAb fully abolished the RS-induced LS revealed by UMB in female, but partially reduced it in male mice. (A and C) Tactile frequency of response at baseline (0) was collected in (A) female and (C) male mice followed by three consecutive episodes of RS for 2 h, each. CA was assessed on indicated days after the first injection over a time course of 16 days. On Day 14, 48 h before the UMB inhalational exposure, mice were treated intraperitoneally with the anti-CGRP mAb, fremanezumab, at 30 mg/kg or control. (B and D) On Day 16, baseline (0) response was collected, followed by inhalational exposure of UMB (0.01M/500 µl, each) with measurements collected hourly for 5 h. The anti-CGRP mAb treatment fully abolished in (B) female and partially reduced in (D) male mice the RS-induced LS, revealed by lack of effect of UMB exposure on eliciting periorbital allodynia. Data are presented as mean ± SEM and analysed using two-way ANOVA followed by Sidak’s (A and B) multiple comparison tests with *P < 0.05 (n = 7–10). Details of statistical analysis are found in Supplementary Table 1.