Horseradish peroxidase (HRP) was injected extracellularly into the auditory nerve of adult mice so that the enzyme could infuse individual spiral ganglion neurons. Forty-two well-stained neurons were reconstructed through serial sections from their cell bodies to peripheral terminations in the organ of Corti with the aid of a light microscope and drawing tube. No neuron was observed to innervate both inner and outer hair cells (IHCs and OHCs). Previous observations from neonatal mammals that reported that IHCs and OHCs were innervated by the same neuron are thus presumed to describe a transient developmental phenomenon. Two populations of spiral ganglion neurons were determined on the basis of the differences in receptor innervation. The type I neurons innervated exclusively IHCs by way of thick (1-2 microns) radial fibers, whereas the type II neurons innervated only OHCs by way of thin (approximately 0.5 micron) outer spiral fibers. Certain features of the peripheral process in the vicinity of the cell body were highly correlated with fiber type. This pattern of separate innervation of IHCs and OHCs by type I and type II neurons, respectively, may represent the general plan of afferent organization for the adult mammalian cochlea.

Previous attempts to trace the central pathways of the thin axons from type II spiral ganglion neurons have been hampered by technical difficulties such as fading of the reaction product as distance increases from the injection site (Ryugo et al.: Soc. Neurosci. Abstr. 12:779, '86; Brown: J. Comp. Neurol. 260:591-604, '87). By using small rodents (gerbils and mice), which have short auditory nerves, we have succeeded in filling the entire central axon and terminals of type II neurons after peripheral injections of horseradish peroxidase. The general course of the type II fibers within the auditory nerve and cochlear nucleus is similar to that of type I fibers except that terminals from type II neurons are often found in regions of the cochlear nucleus that have high densities of granule cells.

The spiral ganglia of the cat, gerbil, mouse, rat, and human were immunohistochemically stained with various monoclonal neurofilament antibodies. Three antibodies to the 200-kD neurofilament protein (R-3, Dräger et al., '84; ICN anti-200, clone NE14, Debus et al., '83; RT-97, Wood and Anderton, '81) labeled the somata of type II spiral ganglion neurons but not those of type I ganglion neurons. In the extreme base of the cochlea of cats, mice and rats, there was intense labeling of a few (less than 0.5% of the total ganglion population) large neurons resembling type I ganglion neurons. Several other neurofilament antibodies (Amersham anti-68, Amersham and ICN anti-160, and SMI-32) did not specifically label type II ganglion neurons but instead labeled all neurons of the spiral ganglion. These two patterns of labeling prompted us to investigate the cause for this difference. Because antibodies against the 200-kD neurofilament protein preferentially labeled type II neurons and because 200-kD neurofilament is highly phosphorylated, we treated cochlear tissue with alkaline phosphatase in order to remove phosphate groups. This treatment eliminated the intense labeling of type II neurons with R-3, ICN anti-200, and RT-97, but had no effect on the intense labeling of ganglion cell bodies observed with the other neurofilament antibodies tested. This evidence suggests that labeling occurs because of the cytoplasmic presence of phosphorylated 200-kD neurofilament protein in type II ganglion neurons. Populations of neurons may thus differ in their neurofilament epitopes and monoclonal antibodies can be used to mark such differences.