Spinal Mechanisms of Testosterone Involved in Itch Regulation

ABSTRACT Background and Objective Clinical and epidemiological data demonstrate that females exhibit higher itch sensitivity than males, and prior research has confirmed estrogen exacerbates pruritus to drive this sexual dimorphism. Nevertheless, it remains poorly defined whether testosterone, the primary androgen, exerts anti‐pruritic effects and contributes to sex disparities in itch perception. This study therefore has three primary objectives: (1) to characterize testosterone's regulatory effects on acute and chronic itch across male and female mice; (2) to identify spinal androgen receptor‐expressing neuronal populations mediating testosterone's anti‐pruritic actions; (3) to elucidate the downstream GABAergic synaptic cascade by which testosterone suppresses spinal itch transmission. Methods C57BL/6 mice and GRPR‐GFP mice were used in this study. Behavioral tests were performed to evaluate the effects of exogenous testosterone administration, orchiectomy, androgen receptor (AR) antagonist flutamide, and GABA A receptor (GABA A R) antagonist bicuculline on acute itch induced by histamine or chloroquine and chronic itch induced by 2,4‐dinitrofluorobenzene (DNFB), as well as anxiety‐like behaviors assessed by elevated plus‐maze (EPM) and open‐field test (OFT). Double immunofluorescence staining was applied to detect the co‐localization of AR and PAX2 in the spinal dorsal horn. Whole‐cell patch‐clamp electrophysiology was used to record synaptic transmission and excitability of GRPR + neurons in spinal cord slices. Results Testosterone contributed to sex differences in itch in mice. Exogenous testosterone suppressed histamine‐ or chloroquine‐evoked acute itch and alleviated DNFB‐induced chronic itch and anxiety‐like behaviors. Orchiectomy enhanced itch sensitivity, which was reversed by testosterone replacement. AR was predominantly expressed on PAX2 + GABAergic inhibitory interneurons in the superficial spinal dorsal horn. Testosterone increased spontaneous inhibitory synaptic input and reduced excitability of GRPR + neurons. The antipruritic effect of testosterone was blocked by intrathecal flutamide or bicuculline. Conclusion Testosterone inhibits itch transmission at the spinal level by activating AR on GABAergic interneurons, enhancing GABA release, and subsequently suppressing GRPR + neurons via GABA A R. Testosterone acts as an endogenous antipruritic hormone and contributes to the sex difference in itch perception, providing a novel target for clinical itch treatment.