Mapping of uterine-related neurons in central nervous system of mice by trans-synaptic tracing with pseudorabies virus

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The uterus, a key organ for maintaining reproductive health and fertility, is precisely regulated by central nervous system (CNS). This study employed multimodal neural tracing and c-Fos staining to investigate the multisynaptic pathways connecting uterus and CNS in mice, as well as the potential neural mechanisms through which olfactory stimuli influence uterine activity. First, the PRV-CAG-EGFP was injected into uterine myometrium. PRV-infected neurons were observed to ascend over time from spinal cord and medulla oblongata to forebrain, ultimately appearing in regulatory nuclei such as paraventricular nucleus of hypothalamus (PVN), medial amygdala (MeA) and central amygdala (CeA), confirming the multilevel neural connections from uterus to CNS. Further anatomical tracing identified connection between olfactory/accessory olfactory bulbs and MeA, but not CeA, suggesting that MeA may act as a crucial relay for olfactory information transmission. In odorant screening, butyric acid (BA) activated olfactory bulb (OB), PVN, MeA, and CeA. This effect was abolished following nasal mucosal injury, indicating its dependence on an intact olfactory pathway. Additionally, BA also activated a subset of oxytocin-positive neurons within PVN. Co-labeling experiments combining PRV tracing and c-Fos staining identified neurons in PVN, CeA, and MeA that were both activated by BA and trans -synaptically connected to uterus. Notably, within PVN, some of these neurons co-expressed oxytocin (OT), c-Fos, and PRV. In summary, this study preliminarily proposed a potential uterus-brain neural circuit and suggests that olfactory cues may influence uterine activity via activation of circuit-specific neurons linked to uterus, including oxytocinergic populations. These findings provide experimental insight into the neural mechanisms underlying odor-mediated modulation of visceral function. • Uterus-to-CNS multisynaptic pathway mapped via retrograde PRV tracing. • Medial amygdala identified as a key olfactory relay for uterine modulation. • Butyric acid activates oxytocin neurons in PVN linked transsynaptically to uterus.

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