Nociceptors are functionally male or female: from mouse to monkey to man

Abstract

The prevalence of many pain conditions often differs between sexes. In addition to such quantitative distinctions, sexual dimorphism may also be qualitative reflecting differences in mechanisms that promote pain in men and women. A major factor that influences the likelihood of pain perception is the threshold for activation of nociceptors. Peripheral nociceptor sensitization has been demonstrated to be clinically relevant in many pain conditions. Whether peripheral nociceptor sensitization can occur in a sexually dimorphic fashion, however, has not been extensively studied. To address this fundamental knowledge gap, we used patch clamp electrophysiology to evaluate the excitability of dorsal root ganglion neurons from male or female rodents, non-human primates, and humans following exposure to putative sensitizing agents. Previous studies from our laboratory, and others, have shown that prolactin promotes female-selective pain responses in rodents. Consistent with these observations, dorsal root ganglion neurons from female, but not male, mice were selectively sensitized by exposure to prolactin. The sensitizing action of prolactin was also confirmed in dorsal root ganglion neurons from a female macaque monkey. Critically, neurons recovered from female, but not male, human donors were also selectively sensitized by prolactin. In the course of studies of sleep and pain, we unexpectedly observed that an orexin antagonist could normalize pain responses in male animals. We found that orexin B produced sensitization of male, but not female, mouse, macaque, and human dorsal root ganglion neurons. Consistent with functional responses, increased prolactin receptor and orexin receptor 2 expression was observed in female and male mouse dorsal root ganglia, respectively. Immunohistochemical interrogation of cultured human sensory neurons and whole dorsal root ganglia also suggested increased prolactin receptor expression in females and orexin receptor 2 expression in males. These data reveal a functional double dissociation of nociceptor sensitization by sex, which is conserved across species and is likely directly relevant to human pain conditions. To our knowledge, this is the first demonstration of functional sexual dimorphism in human sensory neurons. Patient sex is currently not a common consideration for the choice of pain therapy. Precision medicine, based on patient sex could improve therapeutic outcomes by selectively targeting mechanisms promoting pain in women or men. Additional implications of these findings are that the design of clinical trials for pain therapies should consider the proportions of male or female patients enrolled. Lastly, re-examination of selected past failed clinical trials with subgroup analysis by sex may be warranted.

Keywords: orexin; pain; peripheral nociceptor sensitization; prolactin; sexual dimorphism.

Figure 1

The hypothalamic peptide prolactin drives sexually dimorphic neuronal hyperexcitability in rodent sensory neurons. (A) Representative action potential traces from female dorsal root ganglion (DRG) neurons evoked at the 200 pA current step after overnight treatment with 50 nM mouse prolactin (mPRL). (B) mPRL induces hyperexcitability across a range of current injection pulses and inset shows the 200 pA step. (C) Rheobase, the minimum current injection required to fire a single action potential, and (D) the resting membrane potential (RMP) were not affected by treatment with mPRL. (E) Representative traces of male mouse DRG neurons showing that mPRL did not affect (F) excitability, (G) rheobase or (H) the RMP of these neurons. (I) Representative western blots illustrating levels of PRL receptor-long isoform (PRLR-L) and actin in naïve mice (n = 5 females and 5 males). (J) Quantification of PRLR-L expression, which was only detectable in female mice. Data shown as mean ± standard error of the mean. Number of samples as indicated in the figure. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 20 mV and 200 ms. Statistical comparisons are shown in Supplementary Table 2.

Figure 2

Divergent prolactin receptor expression drives neuronal hyperexcitability following prolactin treatment in female human sensory neurons. (A) Representative action potential traces evoked at 1500 pA after overnight incubation with 50 nM human prolactin (hPRL), which (B) increased excitability of female sensory neurons. Inset shows the 1500-pA current step. (C) hPRL treatment decreased rheobase. (D) Resting membrane potential (RMP) was not affected. (E) Representative traces from male human sensory neurons, which (F) did not display changes in excitability. Inset shows the 1500-pA current step. (G) Rheobase and (H) resting membrane potential were unchanged. (I) Representative immunostaining for PRL receptor (PRLR) and the neuronal marker NeuN in cultured human sensory neurons from female and male donors. Robust expression of PRLR in dorsal root ganglia (DRG) was observed in cells from female donors (top row). Cultured human sensory neurons from males demonstrated markedly lower PRLR expression (bottom row). White arrows mark regions of significant overlap between NeuN and PRLR staining in the somatic compartment. Scale bars = 50 µm. Data shown as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 20 mV and 200 ms (A and E). Number of cells recorded indicated in the figure. Statistical details are listed in Supplementary Table 2.

Figure 3

Treatment with orexin b induces hyperexcitability and Hcrtr2 is expressed at higher levels selectively in male rodent sensory neurons. (A) Representative traces from female dorsal root ganglion (DRG) neurons after treatment with 180 nM orexin B (ORXB) evoked at the 200 pA current step. (B) Treatment with ORXB did not affect the excitability, (C) the rheobase or (D) the resting membrane potential (RMP) in these neurons. (E) Representative traces from male DRG neurons treated with ORXB at the 200 pA current step. (F) Excitability was dramatically increased following treatment with ORXB in male sensory neurons and the inset shows the 200 pA current step. (G) ORXB treatment decreased the rheobase, but (H) did not affect the RMP in male mouse sensory neurons. (I) Representative western blots illustrating levels of Hcrtr2 and actin in naïve mice (n = 5 females and 5 males). (J) Quantification of Hcrtr2 expression, which was characterized by high levels of expression in male mice relative to female mice. Data shown as mean ± standard error of the mean. The number of samples is indicated in the figure. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 20 mV and 200 ms. Statistical comparisons are shown in Supplementary Table 2.

Figure 4

Hcrtr2 is expressed in male human sensory neurons and treatment with orexin b induces hyperexcitability. (A) Representative action potential traces from female human dorsal root ganglia (hDRG) evoked at 1500 pA. (B) Excitability of female hDRG was not altered by 180 nM orexin B (ORXB) treatment. Inset shows 1500-pA step. (C) Rheobase and (D) resting membrane potential (RMP) were not affected by ORXB treatment. (E) Representative traces evoked at 1500 pA from male hDRG. (F) Excitability of male hDRG increased dramatically after incubation with 180 nM ORXB. Inset shows 1500-pA current step. (G) ORXB reduced the rheobase of male hDRG neurons, but (H) RMP was unchanged. (I) Representative immunostaining for Hcrtr2 and the neuronal marker NeuN in cultured human sensory neurons from female and male donors. No specific staining was detected in cultured female sensory neurons (top row). When investigating cultured male sensory neurons we observed specific staining for Hcrtr2 (bottom row). Scale bars = 50 µm (I). Data are represented as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 20 mV and 200 ms (A and E). The number of cells recorded is indicated in the figure. Statistical details are listed in Supplementary Table 2.