iDISCO+ for the Study of Neuroimmune Architecture of the Rat Auditory Brainstem

Abstract

The lower stations of the auditory system display a complex anatomy. The inner ear labyrinth is composed of several interconnecting membranous structures encased in cavities of the temporal bone, and the cerebellopontine angle contains fragile structures such as meningeal folds, the choroid plexus (CP), and highly variable vascular formations. For this reason, most histological studies of the auditory system have either focused on the inner ear or the CNS by physically detaching the temporal bone from the brainstem. However, several studies of neuroimmune interactions have pinpointed the importance of structures such as meninges and CP; in the auditory system, an immune function has also been suggested for inner ear structures such as the endolymphatic duct (ED) and sac. All these structures are thin, fragile, and have complex 3D shapes. In order to study the immune cell populations located on these structures and their relevance to the inner ear and auditory brainstem in health and disease, we obtained a clarified-decalcified preparation of the rat hindbrain still attached to the intact temporal bone. This preparation may be immunolabeled using a clearing protocol (based on iDISCO+) to show location and functional state of immune cells. The observed macrophage distribution suggests the presence of CP-mediated communication pathways between the inner ear and the cochlear nuclei.

Keywords: 4th ventricle; auditory system; choroid plexus; cochlear nucleus; iDISCO+; inner ear; macrophage; microglia.

Figure 1

Inner ear. The fluorescence signal originates both from Iba-1 labeling and autofluorescence. Panels (A,C,D) show maximum projections from Z-stacks (A: 10 μm; B: 50 μm; D: 20 μm). (A) Representative section of the cochlea. (B) 3D reconstruction of the inner ear viewed from the medial side. (C) Higher magnification detail of a single cochlear turn. Same parameters as panel (A). (D) Longitudinal section of ED and ES (pseudocolored blue) displaying macrophage populations. The ED follows CC and emerges at the inner surface of the bone as ES. A dural fold (asterisk) continuous with the periosteum separates ES from CP. AN, auditory nerve; BM, basilar membrane; CA, cochlear aqueduct; CC, crus commune; CN, cochlear nucleus; CO, cochlea; CP, choroid plexus; ED, endolymphatic duct; ES, endolymphatic sac; MV, modiolar vein; OC, organ of corti; RM, reissner membrane; SC, semicircular canal; SG, spiral ganglion; SL, spiral ligament; SLi, spiral limbus; SV, stria vascularis.

Figure 2

Hindbrain. (A) Sagittal z-projection of rat hindbrain showing the lateral exit of the 4th ventricle, partially covered by a meningeal fold (asterisk), pseudocolored with a Fire LUT (color corresponds to Z-depth relative to initial section). Inset: 3D model showing the relative positions of CN, 4th ventricle and flocculus. (B) Coronal optical section showing CP spatial relations at the DCN caudal end. Inset: 3D model (posterolateral view) showing CN position (blue) relative to ventricle (gray) and flocculus (beige). (C) Higher magnification sagittal optical section showing a CP branch associated to the DCN surface. (D) Segmented 4th ventricle volume (red), showing holes corresponding to CP-DCN contacts. CN model (blue; Muniak et al., 2013) is shown to explain the “virtual mold” shape features. (E) DCN vascular labeling. Z-stack of 500 optical sections (500 μm), pseudocolored as panel (A). (E′) CN model shows the region (left of clipping plane) and stack direction (blue arrow) of panel (E). (F) CP labeling with IgG outlines stroma rather than vessel lumen, reaching up to epithelial tight junctions, giving the CP a “spiny” appearance in single optical sections. (F′) Z-projection of CP, pseudocolored as panel (A). 3D models are from mouse brain, and for qualitative reference only, since rat structures are similar but not identical.