M. Christian Brown

Role of α9 Nicotinic ACh Receptor Subunits in the Development and Function of Cochlear Efferent Innervation

Cochlear outer hair cells (OHCs) express alpha9 nACh receptors and are contacted by descending, predominately cholinergic, efferent fibers originating in the CNS. Mice carrying a null mutation for the nACh alpha9 gene were produced to investigate its role(s) in auditory processing and development of hair cell innervation. In alpha9 knockout mice, most OHCs were innervated by one large terminal instead of multiple smaller terminals as in wild types, suggesting a role for the nACh alpha9 subunit in development of mature synaptic connections. Alpha9 knockout mice also failed to show suppression of cochlear responses (compound action potentials, distortion product otoacoustic emissions) during efferent fiber activation, demonstrating the key role alpha9 receptors play in mediating the only known effects of the olivocochlear system.

Projections of thin (type-II) and thick (type-I) auditory-nerve fibers into the cochlear nucleus of the mouse.

Injections of horseradish peroxidase into the mouse spiral ganglion were used to label type-I and type-II afferent fibers. Axons presumed to be from type-II spiral ganglion cells because of their small diameter (less than 0.7 microns) and lack of nodes of Ranvier were traced to their terminations in the cochlear nucleus. Thicker fibers presumed to be from type-I ganglion cells were also reconstructed. Type-I and type-II axons labeled by basal turn injections bifurcate together in the dorsal part of the auditory nerve root, forming a branch that ascends into the anteroventral cochlear nucleus and a branch that descends into the posteroventral cochlear nucleus. Type-I fibers formed many collaterals ending in terminal swellings whereas type-II fibers were almost unbranched. Swellings from type-I and type-II fibers were often formed alongside one another. Examples of this proximity include terminal swellings of root collaterals in the auditory nerve root, as well as type-II en passant swellings and type-I terminal swellings throughout the ventral cochlear nucleus. The projections are dissimilar, however, since every type-II fiber projects at least one collateral to the granule-cell lamina. These collaterals usually end in neuropil forming the border between the ventral cochlear nucleus and the granule-cell lamina. In this border region, the type-II terminals overlap with those of branches from thick axons of the olivocochlear (efferent) bundle. Type-II fibers also differ from type-I fibers by only rarely coursing into the dorsal cochlear nucleus and by forming very few terminal swellings. En passant swellings, however, are numerous on type-II fibers, with ellipsoidal-shaped swellings prominent in the nerve root, and angular and complex-shaped swellings common nearer the terminals. We suggest that the latter swellings are associated with type-II synapses whereas the ellipsoidal swellings represent non-synaptic structures.

Medial Olivocochlear Reflex Interneurons Are Located in the Posteroventral Cochlear Nucleus: A Kainic Acid Lesion Study in Guinea Pigs

The medial olivocochlear (MOC) reflex arc is probably a three‐neuron pathway consisting of type I spiral ganglion neurons, reflex interneurons in the cochlear nucleus, and MOC neurons that project to the outer hair cells of the cochlea. We investigated the identity of MOC reflex interneurons in the cochlear nucleus by assaying their regional distribution using focal injections of kainic acid. Our reflex metric was the amount of change in the distortion product otoacoustic emission (at 2f1–f2) just after onset of the primary tones. This metric for MOC reflex strength has been shown to depend on an intact reflex pathway. Lesions involving the posteroventral cochlear nucleus (PVCN), but not the other subdivisions, produced long‐term decreases in MOC reflex strength. The degree of cell loss within the dorsal part of the PVCN was a predictor of whether the lesion affected MOC reflex strength. We suggest that multipolar cells within the PVCN have the distribution and response characteristics appropriate to be the MOC reflex interneurons. J. Comp. Neurol. 487:345–360, 2005.

Postsynaptic Targets of Type II Auditory Nerve Fibers in the Cochlear Nucleus

Type II auditory nerve fibers, which provide the primary afferent innervation of outer hair cells of the cochlea, project thin fibers centrally and form synapses in the cochlear nucleus. We investigated the postsynaptic targets of these synapses, which are unknown. Using serial-section electron microscopy of fibers labeled with horseradish peroxidase, we examined the border of the granule-cell lamina in mice, an area of type II termination that receives branches having swellings with complex shapes. About 70% of the swellings examined with the electron microscope formed morphological synapses, which is a much higher value than found in previous studies of type II swellings in other parts of the cochlear nucleus. The high percentage of synapses enabled a number of postsynaptic targets to be identified. Most of the targets were small dendrites. Two of these dendrites were traced to their somata of origin, which were cochlear-nucleus “small cells” situated at the border of the granule-cell lamina. These cells did not appear to receive any terminals containing synaptic vesicles that were large and round, indicating a lack of input from type I auditory nerve fibers. Nor did type II swellings or targets participate in the synaptic glomeruli formed by mossy terminals and the dendrites of granule cells. Other type II synapses were axosomatic and their targets were large cells, which were presumed multipolar cells and one cell with characteristics of a globular bushy cell. These large cells almost certainly receive additional input from type I auditory nerve fibers, which provide the afferent innervation of the cochlear inner hair cells. A few type II postsynaptic targets—the two small cells as well as a large dendrite—received synapses that had accompanying postsynaptic bodies, a likely marker for synapses of medial olivocochlear branches. These targets thus probably receive convergent input from type II fibers and medial olivocochlear branches. The diverse nature of the type II targets and the examples of segregated convergence of other inputs illustrates the synaptic complexity of type II input to the cochlear nucleus.

Anatomy of Olivocochlear Neurons

Hair cell receptors for the hearing and balance organs, and the lateral line, are unique among the senses by receiving an efferent innervation of the periphery. Olivocochlear (OC) neurons supply this efferent innervation, and they are the most peripheral of the many descending neural systems of the central auditory pathway (see Schofield, Chap. 9). OC neurons are named by their origins in the superior olivary complex and terminations in the cochlea (Fig. 2.1). In the cochlea, they innervate the hair cells and auditory-nerve fibers. This chapter mainly covers the new ground on OC anatomy in mammals since Warr’s (1992) comprehensive chapter on this topic about 15 years ago. Since that time, there is even stronger evidence for the separate innervation of the periphery by the two major groups of OC neurons. It is also now clear that both of these groups consist of distinct subgroups. There is additional information on the reflex pathways leading up to OC neurons that enables their response to sound. Overall, this anatomy may help to define the functions that OC neurons perform in the sense of hearing.

Planar multipolar cells in the cochlear nucleus project to medial olivocochlear neurons in mouse

Medial olivocochlear (MOC) neurons originate in the superior olivary complex and project to the cochlea, where they act to reduce the effects of noise masking and protect the cochlea from damage. MOC neurons respond to sound via a reflex pathway; however, in this pathway the cochlear nucleus cell type that provides input to MOC neurons is not known. We investigated whether multipolar cells of the ventral cochlear nucleus have projections to MOC neurons by labeling them with injections into the dorsal cochlear nucleus. The projections of one type of labeled multipolar cell, planar neurons, were traced into the ventral nucleus of the trapezoid body, where they were observed terminating on MOC neurons (labeled in some cases by a second cochlear injection of FluoroGold). These terminations formed what appear to be excitatory synapses, i.e., containing small, round vesicles and prominent postsynaptic densities. These data suggest that cochlear nucleus planar multipolar neurons drive the MOC neurons response to sound. J. Comp. Neurol. 520:1365–1375, 2012.

Olivocochlear Neuron Central Anatomy Is Normal in α9 Knockout Mice

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the α9 nicotinic acetylcholine receptor subunit. In this α9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse’s apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in α9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

Ultrastructure of synaptic input to medial olivocochlear neurons

Medial olivocochlear (MOC) neurons project from the brain to the cochlea to form the efferent limb of the MOC reflex. To study synaptic inputs to MOC neurons, we retrogradely labeled these neurons using horseradish peroxidase injections into the cochlea. Labeled neurons were identified in the ventral nucleus of the trapezoid body and documented with the light microscope before being studied with serial‐section electron microscopy. MOC somata and dendrites were innervated by three different types of synapses, distinguished as either having: 1) large, round synaptic vesicles and forming asymmetric contacts; 2) small, round vesicles plus a few dense core vesicles and forming asymmetric contacts; or 3) pleomorphic vesicles and forming symmetric contacts. The first two types were the most frequent on somata. Acetylcholinesterase‐stained material confirmed that the type containing large, round vesicles is most common on dendrites. We kept track of the synaptic terminals in serial sections and compiled them into three‐dimensional swellings. Swellings with large, round vesicles formed up to seven synapses per swelling, were largest in size, and sometimes formed complex arrangements engulfing spines of MOC neurons. Swellings with small, round vesicles formed up to four synapses per swelling. The morphology of this type of synapse, and the moderate sizes of the swellings forming it, suggests that it originates from posteroventral cochlear nucleus stellate/multipolar neurons. This input may thus provide the sound‐evoked input to MOC neurons that causes their reflexive response to sound. J. Comp. Neurol. 499:244–257, 2006.

Anatomical and Physiological Studies of Type I and Type II Spiral Ganglion Neurons

Primary auditory neurons transmit information from receptor cells in the cochlea, hair cells, to cochlear-nucleus neurons in the brain. The cell bodies of the primary neurons form the spiral ganglion, which is located in the cochlea. Several early studies demonstrated that there was more than one type of spiral ganglion cell (Suzuki et al.,’ 63; Reinecke,’ 67; Kellerhals et al.,’ 67; Merck et al.,’ 77). In pioneering work, Spoendlin (’71,’ 74) showed that most spiral ganglion cells degenerated following section of the auditory nerve. Since the afferent endings on outer hair cells persisted following nerve section, the few remaining “type II” spiral ganglion cells were postulated to innervate the outer hair cells. Using reconstructions of fibers labeled with horseradish peroxidase, Kiang et al. (’82) demonstrated directly that type II neurons sent processes only to outer hair cells whereas type I neurons innervated only the inner hair cells.

Commissural axons of the mouse cochlear nucleus.

The axons of commissural neurons that project from one cochlear nucleus to the other were studied after labeling with anterograde tracer. Injections were made into the dorsal subdivision of the cochlear nucleus in order to restrict labeling only to the group of commissural neurons that gave off collaterals to, or were located in, this subdivision. The number of labeled commissural axons in each injection was correlated with the number of labeled radiate multipolar neurons, suggesting radiate neurons as the predominant origin of the axons. The radiate commissural axons are thick and myelinated, and they exit the dorsal acoustic stria of the injected cochlear nucleus to cross the brainstem in the dorsal half, near the crossing position of the olivocochlear bundle. They enter the opposite cochlear nucleus via the dorsal and ventral acoustic stria and at its medial border. Reconstructions of single axons demonstrate that terminations are mostly in the core and typically within a single subdivision of the cochlear nucleus. Extents of termination range from narrow to broad along both the dorsoventral (i.e., tonotopic) and the rostrocaudal dimensions. In the electron microscope, labeled swellings form synapses that are symmetric (in that there is little postsynaptic density), a characteristic of inhibitory synapses. Our labeled axons do not appear to include excitatory commissural axons that end in edge regions of the nucleus. Radiate commissural axons could mediate the broadband inhibition observed in responses to contralateral sound, and they may balance input from the two ears with a quick time course. J. Comp. Neurol. 521:1683–1696, 2013.