Olivocochlear innervation in the mouse: immunocytochemical maps, crossed versus uncrossed contributions, and transmitter colocalization

Abstract

To further understand the roles and origins of gamma-aminobutyric acid (GABA) and calcitonin gene-related peptide (CGRP) in the efferent innervation of the cochlea, we first produced in the mouse an immunocytochemical map of the efferent terminals that contain acetylcholine (ACh), CGRP, and GABA. Olivocochlear (OC) terminals in inner and outer hair cell (IHC and OHC) regions were analyzed quantitatively along the cochlear spiral via light-microscopic observation of cochlear wholemounts immunostained with antibodies to glutamic acid decarboxylase (GAD), vesicular acetylcholine transporter (VAT), or the peptide CGRP. Further immunochemical characterization was performed in mice with chronic OC transection at the floor of the fourth ventricle to distinguish crossed from uncrossed contributions and, indirectly, the contributions of lateral versus medial components of the OC system. The results in mouse showed that (1) there are prominent GABAergic, cholinergic, and CGRPergic innervations in the OHC and IHC regions, (2) GABA and CGRP are extensively colocalized with ACh in all OC terminals in the IHC and OHC areas, (3) the longitudinal gradient of OC innervation peaks roughly at the 10-kHz region in the OHC area and is more uniform along the cochlear spiral in the IHC area, (4) in contrast to other mammalian species there is no radial gradient of OC innervation of the OHCs, and (5) all OHC efferent terminals arise from the medial OC system and terminals in the IHC area arise from the lateral OC system.

Fig. 1

Glutamic acid decarboxylase (GAD) immunostaining in a cochlear wholemount from the 8-kHz region. A: Brightfield micrograph of the inner spiral bundle (ISB) shows the plexus of GAD positive terminals (e.g., at arrow) beneath inner hair cells in a control mouse. B: Brightfield micrograph of the same cochlear region shown in A demonstrates multiple immunostained terminals beneath almost every outer hair cell (e.g., white arrow). Fascicles of efferent fibers can be seen crossing the tunnel of Corti (black arrow). OHC rows are numbered. C, D: High power Nomarski micrographs of inner and outer hair cell areas, respectively, represent the kind of images used for manual tracing of immunostained terminals and subsequent morphometry. When using objectives with high numerical apertures, the focal plane is thin, and a single micrograph can capture only a small subset of terminals in crisp focus (e.g., white arrows in C and D). When tracing outlines, the focus is rolled to capture each terminal cluster (black arrow in D) at optimal focus. Scale bars in B and D apply to A and C, respectively.

Fig. 2

Innervation densities for terminals positive for vesicular acetylcholine transporter (VAT), calcitonin gene-related peptide (CGRP), and glutamic acid decarboxylase (GAD) in the inner hair cell (IHC) area as a function of cochlear location. For each antibody (see key), the longitudinal gradient of immunopositive terminals in the IHC area was determined for three to four cochleae. Mean values (± standard error of the mean) were obtained by averaging data from similar locations. Innervation densities are expressed as total silhouette area per micrometer of cochlear length. Cochlear position is expressed as a percentage of distance from the base (upper scale) and the frequency correlate, as estimated by a cochlear frequency map (Ehret, 1983).

Fig. 3

Innervation densities for terminals positive for vesicular acetylcholine transporter (VAT), calcitonin gene-related peptide (CGRP), and glutamic acid decarboxylase (GAD) in the outer hair cell (OHC) area as a function of cochlear location. A: Data summed across all three OHC rows. B: Data for each row computed separately. For each antibody (see key), the longitudinal and radial gradients of immunopositive terminals in the OHC area were determined for three to four cochleae. Mean values (± standard error of the mean) were obtained by averaging data from similar locations. Innervation densities are expressed as total silhouette area per OHC.

Fig. 4

Numbers of terminals positive for vesicular acetylcholine transporter (VAT), calcitonin gene-related peptide (CGRP), and glutamic acid decarboxylase (GAD) per outer hair cell (OHC) as a function of cochlear location. Counts of terminals per OHC for the γ-aminobutyric acid, acetylcholine, and CGRP markers were performed at six different cochlear regions in three to four control cochleae. Two additional cochlea were labeled with anti-SNAP25 to estimate the total number of vesiculated (i.e., olivocochlear efferent) terminals per OHC. Values reflect the mean count (± standard error of the mean) of immunopositive terminals for each of the four markers.

Fig. 5

Colocalization of glutamic acid decarboxylase (GAD) and vesicular acetylcholine transporter (VAT) in efferent terminals. Paired images from a cochlea double immunostained with anti-GAD and anti-VAT coupled with fluorescent markers. High and low power micrographs of inner (A, B) and outer (C, D) hair cells are presented. Each pair of micrographs was acquired at the same focal plane, with only the filter set changed. Image intensity was adjusted (the VAT signals were weaker) to facilitate comparison of terminal shapes and sizes.

Fig. 6

Midline section of the olivocochlear (OC) bundle to differentiate medial (MOC) from lateral (LOC) OC projections. A: Schematic cross-section through the brainstem shows locations of LOC and MOC cells, approximate pathways for OC axons from the olivary complex to the cochlea, published values for ipsilateral and contralateral projections of LOC versus MOC systems (Campbell and Henson, 1988; Brown, 1993), and positions of the surgical cuts through the crossed OC bundle (COCB) in the present experiments. UOCB, un-crossed OC bundle. B: Transverse section of an mouse brainstem stained with acetylcholinesterase 10 days after the COCB cut. The black arrow points to a shallow cut made at the floor of the fourth (IVth) ventricle, where the OCB (white arrowhead) decussates. C: Glutamic acid decarboxylase (GAD) immunostaining in a cochlear wholemount from the 8-kHz region 10 days after COCB section. In the inner spiral bundle (ISB), GAD positive terminals form a plexus beneath inner hair cells (black arrow) comparable to that in controls (Fig. 1). However, loss of terminals beneath outer hair cells (OHCs) is obvious: the white arrow points to one of the few remaining immunopositive terminals. A few blood cells appear in the image (black arrow), which stain due endogenous peroxidases. OHC rows are numbered for reference.

Fig. 7

Innervation densities for terminals positive for vesicular acetylcholine transporter (VAT), calcitonin gene-related peptide (CGRP), and glutamic acid decarboxylase (GAD) in outer (OHC) and inner (IHC) hair cell areas after sectioning the crossed olivocochlear bundle (OCB). For each antibody (VAT, CGRP, and GAD), three cochleae were immunostained and analyzed as in controls after a successful crossed OCB section, as determined histologically (see Fig. 6B). The data from control ears (solid symbols) are reproduced for comparison (see Figs. 2-3). The contribution of the crossed OCB (dashed lines) was computed by subtracting the post-cut measures (open symbols) from the control values. Top: Mean (± standard error of the mean) innervation density measured in the IHC region, expressed as total silhouette area per micrometer of cochlear length. Bottom: Mean (± standard error of the mean) innervation density in the OHC region, expressed as total silhouette area per OHC.

Fig. 8

Comparison of anatomic and functional data for the medial olivocochlear (MOC) system. The longitudinal gradients of OC terminals on all three OHC rows (from Fig. 3) were normalized to peak value for each marker. These data were compared with a metric of classic MOC effects on cochlear suppression in the mouse, i.e., the average suppression of compound action potential, expressed as “effective attenuation” in decibels during electrical stimulation of the OC bundle (from Vetter et al., 1999).