Postnatal refinement of auditory nerve projections to the cochlear nucleus in cats

Abstract

Studies of visual system development have suggested that competition driven by activity is essential for refinement of initial topographically diffuse neuronal projections into their precise adult patterns. This has led to the assertion that this process may shape development of topographic connections throughout the nervous system. Because the cat auditory system is very immature at birth, with auditory nerve neurons initially exhibiting very low or no spontaneous activity, we hypothesized that the auditory nerve fibers might initially form topographically broad projections within the cochlear nuclei (CN), which later would become topographically precise at the time when adult-like frequency selectivity develops. In this study, we made restricted injections of Neurobiotin, which labeled small sectors (300-500 microm) of the cochlear spiral ganglion, to study the projections of auditory nerve fibers representing a narrow band of frequencies. Results showed that projections from the basal cochlea to the CN are tonotopically organized in neonates, many days before the onset of functional hearing and even prior to the development of spontaneous activity in the auditory nerve. However, results also demonstrated that significant refinement of the topographic specificity of the primary afferent axons of the auditory nerve occurs in late gestation or early postnatal development. Projections to all three subdivisions of the CN exhibit clear tonotopic organization at or before birth, but the topographic restriction of fibers into frequency band laminae is significantly less precise in perinatal kittens than in adult cats. Two injections spaced > or = 2 mm apart in the cochlea resulted in labeled bands of projecting axons in the anteroventral CN that were 53% broader than would be expected if they were proportional to those in adults, and the two projections were incompletely segregated in the youngest animals studied. Posteroventral CN (PVCN) projections (normalized for CN size) were 36% broader in neonates than in adults, and projections from double injections in the youngest subjects were nearly fused in the PVCN. Projections to the dorsal division of the CN were 32% broader in neonates than in adults when normalized, but the dorsal CN projections were always discrete, even at the earliest ages studied.

Figure 1

A,B. Radial sections through the organ of Corti and adjacent cochlear spiral ganglion at a location 4 mm from the base (≈27 kHz), showing NB injection site in a kitten studied at G63 days (case #4197L). The damage to the developing osseous spiral lamina and a small hemorrhage within Rosenthal's canal are indicated (arrows). Although the somata of a few spiral ganglion neurons appear to be intact (arrowhead), most of the cells have degenerated due to damage from the NB injection. Scale bar in A = 100 μm. Scale bar in B = 50 μm. C. An adult injection site located 6 mm from the base (17 kHz), shows the defect in the osseous spiral lamina through which the injection was made and a small hemorrhage (large arrow). The smaller arrow indicates the profile of the glass pipette tip used to make the injection. A few spiral ganglion cells are still recognizable at the top of the ganglion, but most have degenerated at this location. Scale bar in C = 50 μm. D. Injection site size was determined in serial sections evaluated for evidence of damage to Rosenthal's canal, labeled or damaged spiral ganglion cell somata, the distribution of labeled axons in the osseous spiral lamina and the presence of labeled profiles contacting the inner hair cells. Injections in these experiments labeled spiral ganglion neurons innervating cochlear sectors that averaged 320 μm in kittens and 520 μm in adults, a difference that was statistically significant (Student's t-test, unpaired). Error bars represent standard error of the mean (S.E.M.)

Figure 2

A. Radial section through the organ of Corti at 4 mm from the base (25 kHz) and radial to an injection site in a G64 kitten (case #8026). Numerous intensely labeled auditory nerve fibers are seen passing through the habenula perforata (arrow), with many large labeled terminal profiles contacting the base of the IHC. Note that the tunnel of Corti is open, although it is smaller than in adults. Two types of labeled axons are seen passing to the outer hair region. Upper tunnel crossing fibers (black arrowhead) take the course of adult medial olivocochlear efferent fibers and form large contacts upon the bases of the developing OHC. In addition, many smaller caliber labeled fibers (white arrowhead) are seen crossing at the base of the tunnel close to the basilar membrane and taking a spiral course between the Deiters's cells under the OHCs. The tectorial membrane was removed from this specimen during dissection to facilitate the NB reaction. It should be noted that this image and the one in panel D show unstained, extra-thick (5 μm) plastic sections, which were cut specifically to better illustrate the distribution of labeled fibers within the organ of Corti. These sections lack the sharpness and detail of the semi-thin, toluidine blue stained sections illustrated in panels B,C,E and F. (Scale bar in A= 25 μm and indicates magnification for all micrographs except B and E.) B,C. Radial section from the apical turn (19 mm from the base; 0.7 kHz) in the same cochlea as in A. The organ of Corti is more immature, and although the pillar cells (P) are recognizable, the tunnel of Corti is completely closed. The OHCs are extremely short, the fluid spaces of Nuel have not yet formed around them, and the tectorial membrane is closely apposed to the surface of the organ of Corti with a well-developed marginal pillar (MP) attachment to the Hensons' cells. (Scale bar in B 50 μm and indicates magnification for B and E.) D. Section through the organ of Corti showing labeled fibers in a kitten studied at G63 days (#4197). The section is taken adjacent to a spiral ganglion injection centered 5 mm from the base (≈ 20 kHz). Labeled radial nerve fibers are seen passing through the habenula (arrow), contacting the base of the IHC and passing between the pillar cells (arrowhead) to reach the OHC region, although the tunnel of Corti is still closed throughout the organ of Corti. E, F. Radial section from the apical cochlea (21 mm from the base, ≈500 Hz) in a G62 kitten (case #4167). The spiral limbus, which supports the tectorial membrane in older kittens and adults, is not yet distinct because the inner sulcus has not yet formed. The tectorial membrane is closely apposed to the surface of the organ of Corti and extends only as far as the first row of OHC.

Figure 3

The overall size of the CN was estimated by measuring the total cross-sectional area of the CN in sections cut in the coronal plane at a level just posterior to the entrance of the auditory nerve, as illustrated here in a G64 day kitten (A) and in an adult (B). Scale bar = 0.5 mm. C. The mean CN area was determined for the kitten and the adult groups, and the square root was calculated for each. In the kittens (mean age of coronal cases, 65.5 days), the mean CN area was 1.85 mm² and the square root of this value was 1.36, which was 55.6% of the adult value of 2.46. The difference between the kitten and adults groups was highly statistically significant (Student's t-test, unpaired). Error bars indicate S.E.M.

Figure 4

A. NB labeled auditory nerve fibers in the AVCN projection lamina of a G60 kitten (605) illustrating the immature calyceal endings (arrowheads) which have a simple, spoon-shaped velum. B. NB labeled auditory nerve fibers in the AVCN of a G63 kitten (4197R), showing the greater complexity of the neuropil and the calyceal endings (arrowheads). C. Labeled auditory nerve fibers in the PVCN projection lamina of the same G60 day kitten for which the AVCN is shown in A. (Scale bar in A = 50 μm and indicates magnification for A, B, and C.) D. Fine caliber labeled auditory nerve fibers and terminals in the DCN projection of a G64 kitten (8026L). Scale bar = 50 μm.

Figure 5

Sagittal sections of the CN, showing discrete bands or laminae of central auditory nerve axons labeled by NB injections in the spiral ganglion. The laminae within the AVCN and PVCN are angled relative to each other. Measurements of this angle show that it is significantly narrower or more acute in neonates, as illustrated here in a G60 kitten (A,B), than in adults (C,D). Scale bars = 0.5 mm. Scale bar in B indicates magnification for B and D. E. The mean angle formed by these projections in kittens (average age, 65.5 days) was 61.3 degrees, whereas the mean angle for adult projections was 70 degrees. Error bars indicate S.E.M. The dorsal-to-ventral position of the projection laminae within the VCN depends upon the position (represented frequency) at the injection site. Higher frequency projections are positioned more dorsally and lower frequencies are more ventral in the CN, as illustrated here in the adult CN with 2 separate projection laminae in PVCN and AVCN resulting from injections centered at 35 kHz and 17 kHz in the cochlea (B, D).

Figure 6

CN projection widths were estimated by determining the mean pixel density in windows of 25 pixels (50 μm) across a 400 pixel scan (800 μm) positioned orthogonal to the projection laminae. Scans were always executed beginning at the low frequency side of the lamina(e). A, B. In each image, 3 scans were made orthogonal to each projection lamina. This sagittal section is from a G60 kitten. Scale bar in A = 0.5 mm. Scale bar in B = 0.25 mm. C. The mean pixel density was plotted, as illustrated here for scan b. D. For each lamina, scans from 3 sections (total of 9 scans) were averaged. Threshold level was set by subtracting background density until the first negative value occurred in the window. The average plot was normalized, and projection width was calculated as the distance containing 90% of the total pixel density. This value was 0.18 mm for the AVCN projection illustrated. (This average lamina width is compared to one image of this projection in panel B.)

Figure 7

A. The mean pixel density plot is shown for 20 projection laminae in the AVCN, measured in sagittal sections from 14 CN in 8 kittens. The mean projection width, estimated as the distance containing 90% of the total pixel density, was 210 μm. B. The mean pixel density plot for AVCN projections measured in 3 adults (6 projections) had a 90% width of 250 μm. C. The kitten projections were significantly smaller than the projections in adults. Error bars indicate S.E.M. However, when data were normalized for the smaller size of the CN in kittens, the expected AVCN projection width in kittens was 140 μm. Thus, the AVCN projections in neonates were 53% broader than expected if they were proportionate to the adult projections.

Figure 8

A. Data are shown for PVCN projections in the same 8 kittens for which AVCN data are presented in Figure 7. In one case, however, the projection laminae from 2 injections were fused and thus were omitted from the width measurements. The remaining 18 PVCN projection laminae had a mean width of 190 μm. B. The adult PVCN projections had an average width of 250 μm. C. As in the AVCN, the absolute values for PVCN projections measured in kittens were significantly smaller than those in adults. Error bars indicate S.E.M. But when normalized for the smaller size of the CN in kittens, the expected PVCN projection width was 140 μm, and the measured PVCN projection width in kittens was 36% broader than the adult PVCN projections.

Figure 9

The DCN projection laminae were measured in a separate experimental series sectioned in the coronal plane. A. An exemplary section from the caudal DCN of a G64 day kitten shows 2 projections resulting from injections centered at 2.3 mm and 5.3 mm from the base of the cochlea (36 kHz and 19 kHz, respectively). Scale bar = 0.5 mm and indicates magnification for A and B. B. A similar adult case is illustrated with projections from injections at cochlear locations corresponding to 44 and 27 kHz. C. The normalized mean pixel density plot is shown for total of 13 DCN projections in 10 CN of 8 neonatal animals, which had a mean width of 180μm. Error bars indicate S.E.M. D. A total of 9 projections were measured in 7 CN in 6 adult cats, and these adult DCN projections averaged 230 μm. E. Although the absolute mean values for kitten projections were significantly smaller than those for adults, when normalized for CN size, the expected PVCN projection width for the neonatal kittens was 0.13 mm. Thus, DCN projections in the kittens were 32% broader than the adult projections, when scaled relative to CN size.

Figure 10

A. Mean lamina separation in cases in which double injections were made in a single cochlea. The mean separation between the two resulting projections is shown for the AVCN, the PVCN and the DCN in both adult and neonatal kitten groups. Error bars indicate S.E.M. In the AVCN and PVCN the separation was significantly greater in adults than in kittens. When the adult separation value was normalized for CN size, the calculated expected value was very similar to the actual value measured in neonates for the AVCN, and the 2 values were identical in PVCN. In the DCN the variance was greater, and the mean separation was not significantly different in adults and kittens. B. DCN projections (arrows) resulting from 2 injections placed at about 2.3 and 4.2 mm from the cochlear base (36 and 25 kHz, respectively). Note that the 2 projections are well separated in this G63 subject (4187L), which is representative of the youngest subjects examined. Scale bar = 50 μm.

Figure 11

A. Sagittal section illustrating the VCN projections in one of the youngest kittens, examined at G62 (4177R). The two projection laminae resulting from injections placed about 2 mm apart in the cochlea, centered at about 3.6 and 5.5 mm from the cochlear base (29 and 20 kHz, respectively). Although 2 projections can be distinguished, many intensely labeled axons can be seen distributed throughout the region between the laminae. Scale bar = 0.25 mm. B. In the quantitative analysis of the AVCN projections, the mean pixel density of 9 scans did not return to background level between the peaks representing the 2 projections, suggesting incomplete segregation of the projections. C. In the PVCN of this same case, the 2 projection laminae appeared to be fused, and the average pixel value function for the 9 scans showed only a single peak.

Figure 12

Montage of sagittal sections taken lateral (A) to medial (I) through the CN illustrate the same G62 case shown in Figure 11. Two injections positioned about 2 mm apart in the spiral ganglion resulted in labeled projections that were incompletely segregated in the AVCN and fused in the PVCN. All sections containing labeling in this case are presented. Asterisks indicate the NB-labeled auditory nerve axons entering the CN and distal to the bifurcation into ascending and descending collaterals. Scale bar = 0.5 mm.

Figure 13

Montage of sagittal sections cut lateral to medial (A-I) through the CN in an older kitten studied at G68 (3247L). Two injections placed about 2 mm apart in the cochlea resulted in 2 distinct CN projection laminae that were well-separated in both AVCN and PVCN. Sections are shown at the same magnification as those in Figure 12, but in this series 5 of the 14 sections containing labeled projections were omitted in order to fit the illustration on the page due to the significant increase in the size of the CN in this older kitten. Asterisks indicate the labeled auditory nerve axons entering the CN. Scale bar = 0.5 mm.