Abstract
The extraorbital lacrimal glands (ELGs) secret tears to maintain a homeostatic environment for ocular surfaces, and pheromones to mediate social interactions. Although its distinct gender-related differences in mice and rats have been identified, its comprehensive histology together with whole-brain neuronal network remain largely unknown. The primary objective of the present study was to investigate whether sex-specific differences take place in histological and physiological perspectives. Morphological and histological data were obtained via magnetic resonance imaging (MRI), hematoxylin-eosin (HE) staining in mice and rats of both genders. The innervating network was visualized by a pseudorabies virus (PRV) mediated retrograde trans-multi-synaptic tracing system for adult C57BL6/J mice of both genders. In terms of ELGs' anatomy, mice and rats across genders both have 7 main lobes, with one exception observed in female rats which have only 5 lobes. Both female rats and mice generally have relatively smaller shape size, absolute weight, and cell size than males. Our viral tracing revealed a similar trend of innervating patterns antero-posteriorly, but significant gender differences were also observed in the hypothalamus (HY), olfactory areas (OLF), and striatum (STR). Brain regions including piriform area (Pir), post-piriform transition area (TR), central amygdalar nucleus (CEA), medial amygdalar nucleus (MEA), lateral hypothalamic area (LHA), parasubthalamic nucleus (PSTN), pontin reticular nucleus (caudal part) (PRNc), and parabrachial nucleus, (PB) were commonly labeled. In addition, chemical isotope labeling-assisted liquid chromatography-mass spectrometry (CIL-LC-MS) and nuclear magnetic resonance spectroscopy (NMR spectroscopy) were performed to reveal the fatty acids and metabolism of the ELGs, reflecting the relationship between pheromone secretion and brain network. Overall, our results revealed basic properties and the input neural networks for ELGs in both genders of mice, providing a structural basis to analyze the diverse functions of ELGs.
Highlights
The extraorbital lacrimal glands (ELGs) are the tubulo-acinar exocrine glands located below the outer ear in the subcutaneous tissue (Šemanjski et al, 2021)
To identify the whole-brain neurons innervating ELGs, C57BL6/J mice and Pseudorabies virus (PRV)-based retrograde transmulti-synaptic tracing system were utilized with simplified experimental procedures along with PRV tracing principle shown in Figure 1. 4.5 days of PRV transmission was employed to best visualize the whole brain pattern of infection based on our initial experimental setup, which has 2.5 d PRV transmission with infection only reaching up to the level of brainstem and medulla oblongata even with longer IHC antibody incubation
The present study elucidated whole-brain direct innervations to ELGs in both genders of rats and mice, undertaking comprehensive histological visualization together with a detailed analysis of the free fatty acids (FFAs) secreted from the ELGs
Summary
The extraorbital lacrimal glands (ELGs) are the tubulo-acinar exocrine glands located below the outer ear in the subcutaneous tissue (Šemanjski et al, 2021). ELGs are well-known for their role in the maintenance of the homeostatic microenvironment for ocular surfaces by tear secretion, with less attention given to the importance of social behavior regulation and emotional process (Hirayama et al, 2013). Several previous studies have identified the pheromonal role of the exocrine gland-secreting peptides (ESPs) in the tear fluid that are secreted by mice ELGs, which modulate sexual communication between rodents (Cavaliere et al, 2014). Intraspecies communication via pheromones plays an important role in social and sexual behaviors, which are crucial for survival and reproduction in animals (Tirindelli et al, 2009). The innervation patterns of ELGs in the brain and the exclusively neural basis of the complex interplay between the brain and the ELGs have not been mapped in detail to date
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