Up-regulation of GAP-43 in the chinchilla ventral cochlear nucleus after carboplatin-induced hearing loss: correlations with inner hair cell loss and outer hair cell loss

Abstract

Inner ear damage leads to nerve fiber growth and synaptogenesis in the ventral cochlear nucleus (VCN). In this study, we documented the relationship between hair cell loss patterns and synaptic plasticity in the chinchilla VCN using immunolabeling of the growth associated protein-43 (GAP-43), a protein associated with axon outgrowth and modification of presynaptic endings. Unilateral round window application of carboplatin caused hair cell degeneration in which inner hair cells (IHC) were more vulnerable than outer hair cells (OHC). One month after carboplatin treatment (0.5-5 mg/ml), we observed varying patterns of cochlear hair cell loss and GAP-43 expression in VCN. Both IHC loss and OHC loss were strongly correlated with increased GAP-43 immunolabeling in the ipsilateral VCN. We speculate that two factors might promote the expression of GAP-43 in the VCN; one is the loss of afferent input through IHC or the associated type I auditory nerve fibers. The other occurs when the medial olivocochlear efferent neurons lose their cochlear targets, the OHC, and may as compensation increase their synapse numbers in the VCN.

Figure 1

Photomicrographs of surface preparations of the chinchilla inner ear showing a single row of inner hair cells (IHC, arrows) and three rows of outer hair cells (OHC, arrows). (A) In the untreated control ear IHC and OHC could be clearly recognized with their stereocilia (white arrowheads). Black arrowheads point to a few missing OHC. (B) Thirty-one days after application of a moderate-dose of carboplatin, there was a pronounced loss of hair cells, typically more IHC than OHC. Arrowheads point to empty spaces between remaining IHC. Scale bar = 100 urn.

Figure 2

Expression of GAP-43 in VCN of a naive control animal (A) and in the left and right VCN (contralateraly and ipsilaterally to treatment respectively) of one experimental animal with loss of all hair cells (#8959) 31 days after carboplatin application on right round window (B, C): In the naive control (A), GAP-43 was expressed throughout the VCN at a moderate level in bouton-like structures and some fibers. In contrast, GAP-43 was largely undetectable in the auditory nerve (n8). After carboplatin application on the right round window, which resulted in massive loss of hair cells in the right cochlea, the VCN as well as the auditory nerve showed strong GAP-43 immunoreactivity on the right side, ipsilaterally to treatment (C) while GAP-43 staining on the left side, contralaterally to treatment (B) remained similar to the naive control animal (A). Dashed line in (A) indicates the outline of the VCN. n8, 8th nerve, VCN, ventral cochlear nucleus. Scale bar = 250 urn.

Figure 3

Expression of GAP-43 in the ventral cochlear nucleus (A, B) and auditory nerve (C, D) at high magnification, in one experimental animal (# 8959) 31 days after receiving carboplatin on the right round window, which resulted in loss of IHC and OHC in the treated ear: In VCN on the left side, contralaterally to treatment (A), GAP-43 was present at moderate levels in bouton-like structures (arrowheads). In VCN on the right side, ipsilaterally to treatment (B) GAP-43 was highly expressed in bouton-like structures (arrowheads), in fibers (small arrows) and in ring-like structures (large arrows). Auditory nerve fiber bundles on the left side (C, large arrows) did not contain GAP-43 except for single fibers (small arrow). In contrast, auditory nerve fiber bundles on the right side (D) contained many fibers positive for GAP-43. Scale bar = 100 µm.

Figure 4

Cochleograms showing the degree of hair cell loss in the right ear and photomicrographs showing GAP-43 expression in the right VCN 31 days after carboplatin application on the right round window in four experimental animals: Cochleograms show percent loss of IHC (solid line) and OHC (dashed line), the lower horizontal axis shows the percent distance from the apex; the upper horizontal axis the frequency-place map for the chinchilla cochlea expressed in kHz. (A) Animals with little or no hair cell loss (#8945) showed only moderate GAP-43 expression in VCN, comparable to the naive control. Animals with pronounced hair cell loss showed strong GAP-43 expression in VCN on the same side. (B, C) GAP-43 labeling in the VCN followed a tonotopic manner such that high frequency cochlear lesions resulted in strong GAP-43 labeling in high-frequency regions of the VCN. In animals with partial hair cell loss, there were typically more IHC missing than OHC (#8957 and #8607). These animals also showed a gradient of hair cell loss with more hair cells missing in the basal high frequency region than in the apical low frequency region, as well as stronger GAP-43 expression in the dorsal high frequency VCN than in the ventral low frequency VCN. (D) Animals with total or almost total hair cell loss (#8333) along the entire cochlea showed strong GAP-43 expression along the entire dorsal-to-ventral gradient of the VCN. H, high frequencies; L, low frequencies. Scale bar = 250 µm.

Figure 5

A) Scattergrams showing hair cell loss (IHC, triangles; OHC, squares) plotted against the ipsi-to-contralateral (i:c) GAP-43 staining ratios in the VCN for matching frequencies and animals: Data pairs (N=24) were obtained separately from low-frequency regions (ventral VCN and apical third of cochlea), middle-frequency regions, (middle VCN and middle third of the cochlea) and high-frequency regions (dorsal VCN and basal third of the cochlea). Ratios >1 indicate that GAP-43 expression is greater on the right, treated side relative to the left, untreated side. GAP-43 increased significantly with increases in IHC and OHC losses. (B) Scattergrams for a subset of the data (N=13) covering all cases with little or no loss of OHC (0–24%), but strongly varying amount of IHC loss (0–92%). In this subgroup, GAP-43 expression was correlated with IHC loss but not OHC loss. (C) Scattergrams for a subset of data points (N=13) covering all cases with a high degree of IHC loss (triangles, 79–100%) and strongly varying amounts of OHC loss (1–100%). In this subgroup, the increase in GAP-43 expression was correlated with OHC loss but not IHC loss.