Tyrosine Hydroxylase Expression in Type II Cochlear Afferents in Mice

Abstract

Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The "TH-2A-CreER neurons" radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7-25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea.

Keywords: afferent; cochlea; dopamine; hair cells; hearing; type II fiber.

FIG. 1

Cochlear type II-like neurons express tyrosine hydroxylase (TH). A Apical cochlear turn (P8) of Th ^2a-CreER; Ai3 mice labeled with antibodies to GFP (green). A subset of neuronal somata in the spiral ganglion and projecting fibers are seen. B Antibodies to myosin VI label IHCs and OHCs in same tissue (magenta). C Merge of GFP-myosin VI labels. D, E, F Maximum intensity projections of higher magnification confocal image stacks show GFP-immunopositive fibers crossing the tunnel of Corti to make a basal-ward turn into the spiral bundles beneath OHCs, characteristic for type II fibers. A star (D) marks one of a small fraction of labeled fibers that end near the inner hair cells only in young cochleas. G tdTomato (red) expressed in Th ^2a-CreER; Ai9 cochlea labels type II-like fibers and boutons among OHCs (P25). H Antibody labeling of TH (green) on same tissue. I Merge of tdTomato and TH labels shows overlap of both labels except for the terminal boutons, where TH immunolabel was lower (insets). Scale bar represents 100 μm (A–C), 50 μm (D–F), 10 μm (G–I), and 3 μm for inset.

FIG. 2

TH-2A-CreER does not label type I afferents or efferents. A Spiral ganglion neurons (P30 cochlear apex) in Th ^2a-CreER; Ai3 mice labeled with antibodies to GFP (green) or α3 Na⁺/K⁺ ATPase (magenta-type I afferents) show no overlap between these two types. B Outer hair cell region shows TH-2A-CreER boutons (green) on OHCs, distinct from medial efferent terminals (magenta-α3 Na⁺/K⁺ ATPase). C Spiral ganglion neurons (P30 cochlear apex) in Th ^2a-CreER; Ai3 mice labeled with antibodies to GFP (green) or TuJ1 (red) show no overlap between these two types. D Outer hair cell region shows TH-2A-CreER boutons (green) on OHCs, distinct from TuJ1 labeled medial efferent terminals (red). Dotted ovals indicate position of OHC rows. Scale bar represents 10 μm.

FIG. 3

Some TH-2A-CreER-labeled neurons are peripherin-positive. Organ of Corti (cochlear apex) from P13 Th ^2a-CreER; Ai3 mice immunolabeled with antibodies against GFP (A), and peripherin (B). Arrows indicate type II fibers immunopositive for both GFP and peripherin. Open arrow heads indicate type II fibers expressing peripherin, but not EYFP. Solid arrow heads indicate type II fibers expressing EYFP that appear to be peripherin-negative. C Overlay of GFP (green) and peripherin (red) channels. Images are presented as maximum intensity projections of a stack of confocal micrographs from the apical turn. Panel B is oversaturated posthoc to illustrate relatively weak peripherin signal for illustration. TH-2A-Cre ER labeling is intentionally sparse due to low-dose tamoxifen, so not every type II fiber is EYFP-positive. Peripherin immunolabel was relatively weak; nonetheless, it appears that some TH-2A-Cre ER neurons had very low, or no, peripherin expression (solid arrowheads). Scale bar represents 20 μm.

FIG. 4

Type II afferent morphology changes during development. Apical cochlear turns of Th ^2a-CreER; Ai3 mice were analyzed at P8 (A, C) and P30 (B, D). A Type II afferents (green, anti-GFP) execute right-angle basal-ward turns after crossing the tunnel of Corti at P8. B Type II trajectory (green) is curvilinear at P30. C Type II terminal projections (green) were filamentous at P8. D Enlarged terminal boutons (green) were found on OHCs at P30. While type II fibers ran underneath the three rows of OHCs at younger ages (C, arrow), at older ages, they additionally appeared on the periphery beyond the rows of OHCs (D, arrowhead). Scale bar represents 10 μm in A, B and 5 μm in C, D.

FIG. 5

Branching pattern of an individual type II afferent fiber. Maximum intensity projections of confocal image stacks taken in the cochlear apex of P30 Th ^2a-CreER; Ai3 mice (A). After crossing the tunnel of Corti, type II fibers turn towards the base, sporadically extending a few branches (B, C) before a terminal arborization with 14 terminations (D). B–D Enlargements of minor and major arborizations. Some terminal branches formed “wishbones” with two boutons (arrows). Scale bar represents 10 μm.

FIG. 6

Synaptic structure of type II afferents. Organ of Corti from 3–4-week-old Th ^2a-CreER; Ai3 mice apical cochlear turns were immunolabeled for the presynaptic ribbon protein CtBP2/RIBEYE (red), myosin VI, and GFP. A Type II afferent boutons make synaptic contacts with outer hair cells with or without an associated synaptic ribbon. B Two type II afferent boutons from a single fiber associated with two ribbons. C Three afferent boutons from three different type II afferents making synapses on one OHC. D Magnified image that shows boutons from three fibers. Scale bar represents 1 μm in A, B, D and E and 5 μm in C.

FIG. 7

Transmission electron micrographs and reconstructions of OHC synapses. A–C Single thin (65 nm) sections from three different OHCs (basal turn, FVB/NJ mouse, P21). Scale bar = 1 μm. Large vesiculated efferent terminals cover most of the synaptic pole and lie opposite to a postsynaptic cistern seen at this magnification as a thickening of the hair cell plasma membrane. Smaller non-vesiculated, granular afferents are opposite hair cell ribbons in approximately half the cases. D–F Z-axis projections of 3D reconstructions. A–C provide example single sections from D–F reconstructions. Hair cell membrane in light gray lines, postsynaptic cisterns colored green. Afferent terminals in yellow, olive, or orange. Synaptic ribbons colored red, nearby vesicles turquoise. Sometimes ribbons appear “misplaced,” e.g., lying over the postsynaptic efferent cistern. Abbreviations N nucleus of outer hair cell, E efferent, A afferent, D Deiters’ cell.