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First published online 11 July 2007
doi: 10.1242/dev.001925

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Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

Lin-Chien Huang¹, Peter R. Thorne², Gary D. Housley^1,* and Johanna M. Montgomery^1,

¹ Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
² Section of Audiology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.

View larger version (72K): [in this window] [in a new window]   Fig. 1. Double labeling of TMRD and peripherin distinguished the adult-like innervation pattern of the type I and type II SGN afferent neurite projections within the P6 mouse cochlea. Maximum intensity projection of confocal images demonstrate the independent labeling of the type I and type II SGN by TMRD (red) and peripherin immunofluorescence (green), respectively. TMRD-labeled fibers in the intraganglionic spiral bundles, running around the perimeter of Rosenthal's canal, are consistent with TMRD also labeling the efferent fibers at this mature stage (see Fig. 6). The SGN cell bodies and their associated neurites innervating the hair cells are shown in whole mounts (A) and in cross sections (B,C). (A) Overview of basal turn of the cochlea. (B,C) Details of the sensory hair cell region and the spiral ganglion in the mid-turn of the cochlea. (B) TMRD-labeled neurites innervating the inner hair cells at inner spiral plexus, with additional neurites extending towards outer hair cells. The type I fibers traveled above the peripherin-expressing type II fibers. The latter bypassed the IHC region and traveled along the basilar membrane to form three rows of outer spiral bundles, which innervated the OHCs. (C) TMRD and peripherin immunofluorescence labeled the type I and type II SGNs, respectively. TMRD-labeled neurons were distributed throughout the ganglion, while peripherin-labeled neurons were found close to the intraganglionic spiral bundles. Scale bars: 50 µm in A; 10 µm in B,C. igsb, intraganglionic spiral bundles; ihc, inner hair cells; isp, inner spiral plexus; ohc, outer hair cells; osb, outer spiral bundles.

View larger version (83K): [in this window] [in a new window]   Fig. 2. TMRD and peripherin labeling in the E18 mouse cochlea. (A) An overview of TMRD (red) and peripherin (green) labeling in the cochlea shows that the TMRD-labeled type I neurons were found in both the apical and the mid-turn regions, while peripherin expression in the type II neurons was higher in the mid turn. In this example, peripherin was strongly expressed in a single neuron located close to the modiolus in the apical turn (arrow). (B) TMRD and peripherin labeling was independent in the SGN. The peripherin-expressing neurons were scattered throughout the ganglion. Scale bars: 50 µm in A; 10 µm in B. igsb, intraganglionic spiral bundle; isp, inner spiral plexus; osb, outer spiral bundles.

View larger version (93K): [in this window] [in a new window]   Fig. 3. Immature cochlear afferent innervation pattern at E18 in mice. (A) An overview of TMRD (red; type I SGNs) and peripherin (green; type II SGNs) labeling in a whole-mount preparation displayed as a maximum intensity projection. (B) TMRD- and peripherin-labeled neurites in the organ of Corti exhibited an unorganized innervation pattern under the OHCs. These neurites traveled only a short distance in the outer spiral bundles (OSBs). TMRD labeling was also found under the IHCs. (C,D) Organ of Corti cross section from a single confocal optical section provides an alternative view under the hair cells. (C) TMRD-labeled neurites in the inner spiral plexus region, showing innervation of the IHCs. Type I fibers also traveled along the basilar membrane to innervate the first row of OHCs via the OSBs (arrow). (D) Peripherin-expressing neurites traveled along the basilar membrane, and entered the OSBs to target an OHC. Peripherin-expressing fibers also formed collateral innervation to the IHCs via thin fibers (arrowhead). Scale bars: 50 µm in A; 10 µm in B; 20 µm in C,D. isp, inner spiral plexus; osb, outer spiral bundles.

View larger version (150K): [in this window] [in a new window]   Fig. 4. Development of the afferent innervation in the cochlea during neurite reorganization (P0-P6) in mice. TMRD- and peripherin-labeled neurites undergo refinement (P0-P3) and retraction (P3-P6), as demonstrated by maximum intensity projections of confocal images from cochlear whole-mount preparations. (A,D,G) TMRD labeling (red) of type I afferent nerve fibers. These neurites innervate the IHCs between P0 and P6. Peripherin (green) immunolabeling of type II afferent nerve fibers (B,E,H) and merged images (C,F,I) demonstrate the independence and the relationship between TMRD and peripherin labeling. (A-C) At P0, both TMRD- and peripherin-labeled neurites travel along the OHCs in an unorganized pattern. (D-F) Both TMRD- and peripherin-labeled neurites formed three rows of outer spiral bundles beneath the OHCs in an organized pattern. The inner spiral plexus, associated with the IHCs, exhibited only TMRD labeling, but not peripherin labeling, at P3. (G-I) TMRD labeling was reduced under the OHCs compared with the strong representation of peripherin-expressing neurites in the outer spiral bundle region. Scale bars 10 µm. SGNI, type I spiral ganglion neurons; SGNII, type II spiral ganglion neurons.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Three-dimensional reconstruction of the afferent innervation in the organ of Corti at P3 and P6 in mice. Spatial resolution of the pathways for TMRD- (red) and peripherin- (green) labeled type I and type II neurites, respectively, under the hair cells. (A-C) At P3, both types of afferent nerve fibers were found under the OHCs, but their labeling was independent. TMRD labeling was found in the inner spiral plexus (ISP), forming a calyceal innervation to IHCs. In the outer spiral bundles (OSB) under the OHC, TMRD-labeled neurites were located above peripherin-immunopositive type II neurites in the hair cell region and osseous spiral lamina. (D-F) At P6, TMRD-labeled nerve fibers were largely confined to the ISP, with limited residual projection to the first row of OHCs. At this age, peripherin-labeled neurites formed three prominent rows of OSBs innervating the OHCs, after passing close to the basilar membrane below TMRD-labeled neurites. Scale bars: 10 µm. isp, inner spral plexus; osb, outer spiral bundle; osl, osseous spiral lamina; SGNI, type I spiral ganglion neurons; SGNII, type II spiral ganglion neurons.

View larger version (74K): [in this window] [in a new window]   Fig. 6. TMRD, peripherin and ChAT labeling in the mouse organ of Corti between P0 and P6. Single confocal optical sections overlaid with (A-C,G-I,M-O) and without (D-F,J-L,P-R) transmitted-light images demonstrate the relationship between the labeled afferent and efferent nerve fibers and the cellular structures in the organ of Corti. (A-F) P0 organ of Corti showing peripherin- (green, type II SGNs) and TMRD- (red, type I SGNs) labeled neurites innervate both the IHCs and OHCs; no significant ChAT labeling was detected. (A,D) Peripherin-labeled type II neurites branch to innervate the IHCs, with primary neurite processes passing along the basilar membrane to innervate the three rows of OHCs. (B,E) Type I SGN fiber terminals make close contact with the basal and lateral membrane of the IHCs. (G-L) P3 organ of Corti shows both TMRD and peripherin labeling in the three rows of outer spiral bundles (OSBs); TMRD and ChAT labeling, but not peripherin labeling in the inner spiral plexus (ISP) under the IHCs. (G,J) Peripherin labeling is now absent under the IHCs. (H,K) TMRD labeling is more restricted to the basolateral region of the IHCs compared with P0 (see B,E). (I,L) Weak ChAT signal in the ISP. (M-R) Adult-like primary auditory afferent innervation pattern in the P6 organ of Corti with increased efferent nerve fiber labeling. (M,P) Peripherin-labeled neurites, as for P3, confined to the OSBs. (N,Q) TMRD-labeled neurites are predominantly found under the IHCs, while only minimal labeling was detected in the OSBs. Some TMRD-labeling would be attributed to efferent nerve fibers. (O,R) ChAT immunolabeling in both the ISP and OSBs. Scale bar: 10 µm. bm, basilar membrane; dc, Deiters' cell; isp, inner spral plexus; osb, outer spiral bundle; PPH, peripherin; SGNI, type I spiral ganglion neurons; SGNII, type II spiral ganglion neurons.