Immunohistochemical Identification of Sensory Neuropeptides Calcitonin Gene-Related Peptide, Substance P, and Pituitary Adenylate Cyclase-Activating Polypeptide in Efferent Vestibular Nucleus Neurons

Introduction: The efferent vestibular system (EVS) originates in brainstem efferent vestibular nuclei (EVN) and modifies afferent vestibular signals at their source, in peripheral vestibular organs. Recent evidence suggests that EVS is also involved in the development of motion sickness symptoms, including vertigo and nausea, but the underlying mechanism is unknown. One possible link between EVN and motion sickness symptoms is through the neuropeptide calcitonin gene-related peptide (CGRP). CGRP often co-exists with substance P and pituitary adenylate cyclase-activating polypeptide (PACAP), two neuropeptides with similar vasodilatory effects. Collectively, these sensory neuropeptides have been associated with vestibular migraine pathophysiology and motion sickness. While CGRP and the fast EVS neurotransmitter, acetylcholine (ACh), have previously been identified in EVN neurons and their peripheral terminals, the presence of substance P and PACAP in the EVN has not yet been described.

Methods: We used fluorescent immunohistochemistry combined with confocal microscopy to examine the distribution of these three neuropeptides in the mouse EVN. In transgenic choline acetyltransferase (ChAT)-gCaMP6f mice, EVN neurons were positively identified using the fluorescent expression of gCaMP6f. In wild-type C57/BL6 mice, EVN neurons were confirmed using ChAT immunolabelling.

Results: Consistent with previous studies, CGRP was labelled in a subset of cholinergic EVN neurons. Additionally, we also show evidence for substance P and PACAP expression in EVN of transgenic and wild-type mice.

Conclusion: The presence of CGRP, substance P, and PACAP in EVN neurons suggests a complex peptidergic modulation of cholinergic signalling, whose release into local blood vessels may contribute to motion sickness symptoms.