The Expression and Functional Role of Histamine H3 Receptor in the Mammalian Inner Ear C57BL/6 Mice

Histamine and its receptors have been extensively studied in the context of immune responses, inflammatory processes, and neurotransmitter regulation in the central nervous system. However, their role in the inner ear, particularly within the cochlea, remains poorly understood. The cochlea's ability to convert sound into neural signals relies on the precise coordination between hair cells (HCs) and spiral ganglion neurons (SGNs). Abnormalities in this process during early development or damage acquired later in life can lead to hearing impairment. Given the critical role of the histamine H3 receptor (H3R) in the regulation of synaptic plasticity, neurotransmitter release, and neural signal transmission, investigating its expression and function in the cochlea may provide key insights into the regulatory mechanisms underlying auditory signal processing. In this study, we quantified the relative expression levels of H3R across different cochlear regions using RT-qPCR and further determined its spatial distribution in the postnatal day 4-5 (P4-5) cochlea of C57BL/6 mice via immunofluorescence staining. Additionally, we employed two different H3R antagonists/inverse agonists, ciproxifan and pitolisant, at varying concentrations (10μM, 50μM, and 100μM) to examine their effects on cochlear explants in an in vitro culture system. To exclude the potential cytotoxic effects of the solvent used for H3R antagonists, we also evaluated the impact of dimethyl sulfoxide (DMSO) on HC viability to ensure the reliability of our findings. Our results demonstrated that H3R mRNA expression was significantly higher in the modiolus compared to the organ of Corti and the lateral wall. Immunofluorescence staining further confirmed the presence of H3R in both HCs and SGNs. Treatment with the H3R antagonist ciproxifan for 24 hours induced dose-dependent and region-specific HC damage and loss. In contrast, pitolisant did not induce HC loss but resulted in structural abnormalities in the stereocilia at a high concentration (100μM). Furthermore, the concentrations of DMSO used to dissolve the H3R antagonists were confirmed to have no deleterious effects on cochlear HCs. Both H3R antagonists induced aberrant growth patterns of type II SGN neurites, shifting their projections from the typical basal trajectory toward the cochlear apex. This study shows the expression of H3R in the cochlea of P4-5 C57BL/6 mice, suggesting its potential involvement in cochlear development and function. Specifically, H3R may play a critical role in maintaining the structural integrity and functional stability of HCs and in regulating the neurite guidance and synaptic organization of SGNs. Further investigation into its molecular regulatory mechanisms will be essential for understanding auditory signal processing and developing targeted interventions for hearing impairment.