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The localization of choline acetyltransferase (ChAT) and calcitonin gene-related peptide (CGRP) immunoreactivities in the vestibular end-organs of the guinea pig was examined using immunohistochemistry. A large number of ChAT immunoreactive (ChAT-IR) terminals were found adjacent to the base of vestibular sensory cells. ChAT-IR terminals frequently formed synapses with the chalices of afferent nerves receiving synaptic inputs from type I cells and also formed direct contacts with type II cells. ChAT-IR terminals were filled with small clear vesicles, indicating that they are associated with the vestibular efferent system. All the vestibular efferent terminals were identified to be ChAT immunoreactive by electron microscopy. On the other hand, vestibular efferent terminals did not always show CGRP immunoreactivity. CGRP-immunoreactive (CGRP-IR) efferent terminals frequently made synaptic contacts with the chalices and type II cells. These results suggest that acetylcholine (ACh) and CGRP frequently coexist in the vestibular efferent system and may participate in the regulation of the vestibular function.

In this report we demonstrate the pattern of calcitonin gene-related peptide (CGRP) mRNA and immunoreactivity in the central and peripheral vestibular system of the rat, using a CGRP cRNA probe and a polyclonal CGRP antiserum. We present evidence that somata in all regions of efferent vestibular neurons contain CGRP based on the correspondence between in situ hybridization (mRNA) and immunohistochemistry (mRNA translation product). CGRP immunohistochemistry (CGRPi) and in situ hybridization confirm that CGRPi axons and terminals present in the vestibular neuroepithelium are efferent in origin. Immunoelectron microscopy revealed an extensive innervation of the afferent vestibular pathway by CGRPi terminals that was not limited to the primary afferent chalice, as previously reported by Tanaka et al. (Brain Res 1989;504:31-5). An efferent neuromodulatory role of CGRP can be inferred from the distribution of terminals found on the primary afferent fibers, and type I and type II hair cells. In addition, we present evidence that nicotinic acetylcholine receptor (nAChR) subunit mRNA is expressed by primary afferent cell bodies. On the basis of these data, a hypothetical molecular mechanism of vestibular efferent modulation of the primary afferent pathway is proposed.

The immunocytochemical distribution and coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the rat vestibular endorgans were investigated. SP-like immunoreactivity was found in nervous elements beneath and around hair cells. CGRP-like immunoreactivity was also abundantly distributed beneath and within the sensory epithelia. In the present study, double-staining immunocytochemistry revealed that three different types of immunoreactivities: SP-positive/CGRP-negative, SP-negative/CGRP-positive, and SP/CGRP-positive immunostaining can be distinguished. SP/CGRP-immunoreactive fibers were localized within as well as beneath the sensory epithelia. These fibers often penetrated the epithelia and nearly reached the surface. The present immunocytochemical evidence suggests that different types of peripheral nervous systems may exist in the vestibular periphery.