TRIPLE IMMUNOFLUORESCENCE EVIDENCE FOR THE COEXISTENCE OF ACETYLCHOLINE, ENKEPHALINS AND CALCITONIN GENE-RELATED PEPTIDE WITHIN EFFERENT (OLIVOCOCHLEAR) NEURONS OF RATS AND GUINEA-PIGS

The efferent (olivocochlear) nerve supply to the cochlea is subdivided into a lateral and a medial innervation according to several criteria, e.g. locus of origin in the superior olivary complex and type of synaptic connections established in the organ of Corti. We have used a triple immunofluorescence colocalization approach to determine whether putative cholinergic neurons from the lateral innervation contain both metenkephalin and calcitonin gene-related peptide (CGRP), and whether those from the medial innervation also contain CGRP. About 80% of the choline acetyltransferase (ChAT)-like immunostained lateral efferent neurons within the lateral superior olive were CGRP- and metenkephalin-like immunostained. In the organ of Corti, colocalization of the three antigens within the inner spiral bundle was also found. This bundle contains the lateral efferent synapses, with the dendrites of the primary auditory neurons innervating the sensory inner hair cells. Most of the medial efferent neurons in the ventral nucleus of the trapezoid body were only immunoreactive for ChAT. However, in the rostral part of the nucleus, a minority of ChAT-like immunostained neurons were also CGRP-like immunostained. None of the ChAT-like immunostained medial efferent neurons presented metenkephalin-like immunostaining. In agreement with these brainstem data, partial colocalization of the ChAT- and CGRP-like immunostaining and a lack of metenkephalin immunoreactivity was noted below the sensory outer hair cells, which are the synaptic targets of medial efferent terminals in the organ of Corti. This distinction in the coexistence pattern of the two efferent innervations probably reflects distinct modes of action for acetylcholine in the cochlea. In one case, the effects of acetylcholine on the primary auditory neurons innervating the inner hair cells may require balanced modulation by metenkephalin and CGRP. In the other case, modulation of the effects of acetylcholine on the outer hair cells by neuropeptides would be less critical.