Three cochleas from three cats were used for the ultrastructural analysis. Two cats were from control animals, which, as part of previous physiological studies (Liberman 1978
; Liberman and Beil 1979
), underwent single-fiber recordings from the auditory nerve and had normal responses at all frequency regions [data from MCL94L (Fig. 3 in Liberman 1978
) and MCL109L (Fig. in Liberman 1978
; Liberman and Beil 1979
)]. Another cat, # 93042L, underwent unilateral surgical de-efferentation during the first post-natal week, as described elsewhere (Liberman et al. 2000
). All animal procedures were approved by the IACUC of the Massachusetts Eye and Ear Infirmary. Tissues procured prior to 1980 (when institutional animal care committees were not yet in existence) was done so according to procedures that have subsequently been approved by the local IACUC.
FIG. 3 Morphometry from serial sections shows that terminals contacting OHCs fall into two groups: small, vesicle-poor terminals generated by spiraling dendrites (type-II terminals) and large vesicle-rich swellings traced to radially projecting dendrites (OC (more ...)
All animals were perfused intravascularly with 2.5% glutaraldehyde and 1.5% paraformaldehyde in 0.065M phosphate buffer at pH7.3, followed by intralabyrinthine perfusion of the same fixative and post-fixation at 4°C for several days. After post-fixation, cochleas were perfused with 1% osmium tetroxide for 1h and then rinsed, dehydrated, and embedded in epoxy resins. After polymerization, cochleas were drilled down, and dissected partial turns of the organ of Corti were re-embedded, thinned, and mounted on slides for light-microscopic evaluation. Cochlear lengths were measured by computerized planimetry and a frequency map computed for each ear (Liberman 1982
The completeness of de-efferentation was assessed by measuring the summed diameters of efferent fascicles crossing to the OHC region through the tunnel of Corti using high-N.A. objectives and Nomarski optics. Efferent fibers are distinguished from afferents by their position in the tunnel. These data were obtained in osmium-stained cochlear whole mounts at 12 evenly spaced locations along from the cochlear spiral: Diameter measurements of all tunnel-crossing efferents in one microscopic field from each location were compared among a group of control and surgically de-efferented ears (Liberman et al. 2000
). Previous work showed that this metric is well correlated with the volume of olivocochlear terminals remaining on OHCs (Liberman and Gao 1995
For electron microscopy, selected whole mounts in regions of interest were removed from the slides, trimmed, and mounted on epon blanks for serial sectioning in one of two orientations (Fig. ): parallel to the long axes of OHCs (longitudinal) or parallel to the cuticular plate (horizontal). Section ribbons were harvested on Formvar-coated slot grids, stained with uranyl acetate and lead citrate, and examined either on a Philips CM10 or a JEOL 100CX. For each sectioned block of tissue, a subset of the OHCs in each row was selected for analysis. In MCL94L, blocks from the 0.7- and 1.6-kHz regions were sectioned longitudinally, and micrographs were captured from (1) every (serial) section at an overall magnification of ×29,000 for the 0.7kHz region and (2) every fifth (semi-serial) section at a final magnification of ×12,000–13,500 for the 1.6-kHz region. For MCL109L, multiple blocks were selected to investigate different cochlear regions (i.e., 0.35, 0.75, 1.3, 3.2, 6.3, 12.0, and 23.0kHz). Sections were cut parallel to the reticular lamina, and micrographs were captured from every fifth section at a final magnification of ×12,000–13,500. For the de-efferented ear, 93042L, a block from the 1.0-kHz region was selected, sections were cut horizontally, and micrographs were captured from every fifth section at a final magnification of ×12,000–13,500. For all the section series, neural elements were traced by labeling the photographic montages of each section in the series.
FIG. 1 Schematic illustrating the projections of type-II spiral ganglion cells and medial olivocochlear (OC) neurons in the outer hair cell (OHC) area of a typical mammalian ear, as well as the section planes used for serial-section ultrastructural analysis. (more ...)
For immunohistochemistry, cochleas from CBA/CaJ mice (6–8 weeks) were extracted and fixed in chilled 4% paraformaldehyde for 10 min prior to dissection into half turns without decalcification. Cochlear pieces were blocked for 2h at 4°C in 5% normal horse serum with 1% Triton X-100 and then incubated overnight at 4°C in the primary antibodies (rabbit anti-GluR2/3 from Chemicon at 1:500 and mouse anti-CtBP2 from BD Transduction Labs at 1:200) diluted in 1% normal horse serum with 1% Triton. Secondary antibody incubations followed: the first, overnight at 4°C, included biotinylated donkey anti-mouse (1:200, Jackson ImmunoResearch) plus fluorescent-conjugated chicken anti-rabbit (AlexaFluor 488, Invitrogen, 1:1,000): the second, for 1 h at room temperature, included streptavidin-conjugated AlexaFluor 568 (Invitrogen, 1:1,000) plus a goat anti-chicken conjugated to AlexaFluor 488 (Invitrogen, 1:1,000).