Direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway.

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser. Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible. The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

Optogenetic stimulation of the cochlear nucleus using channelrhodopsin-2 evokes activity in the central auditory pathways.

Optogenetics has become an important research tool and is being considered as the basis for several neural prostheses. However, few studies have applied optogenetics to the auditory brainstem. This study explored whether optical activation of the cochlear nucleus (CN) elicited responses in neurons in higher centers of the auditory pathway and whether it elicited an evoked response. Viral-mediated gene transfer was used to express channelrhodopsin-2 (ChR2) in the mouse CN. Blue light was delivered via an optical fiber placed near the surface of the infected CN and recordings were made in higher-level centers. Optical stimulation evoked excitatory multiunit spiking activity throughout the tonotopic axis of the central nucleus of the inferior colliculus (IC) and the auditory cortex (Actx). The pattern and magnitude of IC activity elicited by optical stimulation was comparable to that obtained with a 50dB SPL acoustic click. This broad pattern of activity was consistent with histological confirmation of green fluorescent protein (GFP) label of cell bodies and axons throughout the CN. Increasing pulse rates up to 320Hz did not significantly affect threshold or bandwidth of the IC responses, but rates higher than 50Hz resulted in desynchronized activity. Optical stimulation also evoked an auditory brainstem response, which had a simpler waveform than the response to acoustic stimulation. Control cases showed no responses to optical stimulation. These data suggest that optogenetic control of central auditory neurons is feasible, but opsins with faster channel kinetics may be necessary to convey information at rates typical of many auditory signals.

Robustness of cortical topography across fields, laminae, anesthetic states, and neurophysiological signal types.

Topographically organized maps of the sensory receptor epithelia are regarded as cornerstones of cortical organization as well as valuable readouts of diverse biological processes ranging from evolution to neural plasticity. However, maps are most often derived from multiunit activity recorded in the thalamic input layers of anesthetized animals using near-threshold stimuli. Less distinct topography has been described by studies that deviated from the formula above, which brings into question the generality of the principle. Here, we explicitly compared the strength of tonotopic organization at various depths within core and belt regions of the auditory cortex using electrophysiological measurements ranging from single units to delta-band local field potentials (LFP) in the awake and anesthetized mouse. Unit recordings in the middle cortical layers revealed a precise tonotopic organization in core, but not belt, regions of auditory cortex that was similarly robust in awake and anesthetized conditions. In core fields, tonotopy was degraded outside the middle layers or when LFP signals were substituted for unit activity, due to an increasing proportion of recording sites with irregular tuning for pure tones. However, restricting our analysis to clearly defined receptive fields revealed an equivalent tonotopic organization in all layers of the cortical column and for LFP activity ranging from gamma to theta bands. Thus, core fields represent a transition between topographically organized simple receptive field arrangements that extend throughout all layers of the cortical column and the emergence of nontonotopic representations outside the input layers that are further elaborated in the belt fields.

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 neuron's response to sound.

Expression studies of osteoglycin/mimecan (OGN) in the cochlea and auditory phenotype of Ogn-deficient mice.

Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds.

Methylthioadenosine phosphorylase (MTAP) in hearing: gene disruption by chromosomal rearrangement in a hearing impaired individual and model organism analysis.

Genes with a role in the auditory system have been mapped by genetic linkage analysis of families with heritable deafness and then cloned through positional candidate gene approaches. Another positional method for gene discovery is to ascertain deaf individuals with balanced chromosomal translocations and identify disrupted or disregulated genes at the site(s) of rearrangement. We report herein the use of fluorescence in situ hybridization (FISH) to map the breakpoint regions on each derivative chromosome of a de novo apparently balanced translocation, t(8;9)(q12.1;p21.3)dn, in a deaf individual. Chromosomal breakpoints were assigned initially by GTG-banding of metaphase chromosomes and then BAC probes chosen to map precisely the breakpoints by FISH experiments. To facilitate cloning of the breakpoint sequences, further refinement of the breakpoints was performed by FISH experiments using PCR products and by Southern blot analysis. The chromosome 9 breakpoint disrupts methylthioadenosine phosphorylase (MTAP); no known or predicted genes are present at the chromosome 8 breakpoint. Disruption of MTAP is hypothesized to lead to deafness due to the role of MTAP in metabolizing an inhibitor of polyamine synthesis. Drosophila deficient for the MTAP ortholog, CG4,802, were created and their hearing assessed; no hearing loss phenotype was observed. A knockout mouse model for MTAP deficiency was also created and no significant hearing loss was detected in heterozygotes for Mtap. Homozygous Mtap-deficient mice were embryonic lethal.

Usherin is required for maintenance of retinal photoreceptors and normal development of cochlear hair cells.

Usher syndrome type IIA (USH2A), characterized by progressive photoreceptor degeneration and congenital moderate hearing loss, is the most common subtype of Usher syndrome. In this article, we show that the USH2A protein, also known as usherin, is an exceptionally large ( approximately 600-kDa) matrix protein expressed specifically in retinal photoreceptors and developing cochlear hair cells. In mammalian photoreceptors, usherin is localized to a spatially restricted membrane microdomain at the apical inner segment recess that wraps around the connecting cilia, corresponding to the periciliary ridge complex described for amphibian photoreceptors. In sensory hair cells of the cochlea, it is associated transiently with the hair bundles during postnatal development. Targeted disruption of the Ush2a gene in mice leads to progressive photoreceptor degeneration and a moderate but nonprogressive hearing impairment, mimicking the visual and hearing deficits in USH2A patients. These data suggest that usherin is required for the long-term maintenance of retinal photoreceptors and for the development of cochlear hair cells. We propose a model in which usherin in photoreceptors is tethered via its C terminus to the plasma membrane and its large extracellular domain projecting into the periciliary matrix, where they may interact with the connecting cilium to fulfill important structural or signaling roles.

Selective removal of lateral olivocochlear efferents increases vulnerability to acute acoustic injury.

Cochlear sensory cells and neurons receive efferent feedback from the olivocochlear (OC) system. The myelinated medial component of the OC system and its effects on outer hair cells (OHCs) have been implicated in protection from acoustic injury. The unmyelinated lateral (L)OC fibers target ipsilateral cochlear nerve dendrites and pharmacological studies suggest the LOC's dopaminergic component may protect these dendrites from excitotoxic effects of acoustic overexposure. Here, we explore LOC function in vivo by selective stereotaxic destruction of LOC cell bodies in mouse. Lesion success in removing the LOC, and sparing the medial (M)OC, was assessed by histological analysis of brain stem sections and cochlear whole mounts. Auditory brain stem responses (ABRs), a neural-based metric, and distortion product otoacoustic emissions (DPOAEs), an OHC-based metric, were measured in control and surgical mice. In cases where the LOC was at least partially destroyed, there were increases in suprathreshold neural responses that were frequency- and level-independent and not attributable to OHC-based effects. These interaural response asymmetries were not found in controls or in cases where the lesion missed the LOC. In LOC-lesion cases, after exposure to a traumatic stimulus, temporary threshold shifts were greater in the ipsilateral ear, but only when measured in the neural response; OHC-based measurements were always bilaterally symmetric, suggesting OHC vulnerability was unaffected. Interaural asymmetries in threshold shift were not found in either unlesioned controls or in cases that missed the LOC. These findings suggest that the LOC modulates cochlear nerve excitability and protects the cochlea from neural damage in acute acoustic injury.

Cochlear efferent feedback balances interaural sensitivity.

Neurons in the lateral superior olive (LSO) compute sound location based on differences in interaural intensity, coded in ascending signals from the two cochleas. Unilateral destruction of the neuronal feedback from the LSO to the cochlea, the lateral olivocochlear efferents, disrupted the normal interaural correlation in response amplitudes to sounds of equal intensity. Thus, lateral olivocochlear feedback maintains the binaural balance in neural excitability required for accurate localization of sounds in space.

Dopaminergic innervation of the mouse inner ear: evidence for a separate cytochemical group of cochlear efferent fibers.

Immunostaining mouse cochleas for tyrosine hydroxylase (TH) and dopamine beta-hydroxylase suggests that there is a rich adrenergic innervation throughout the auditory nerve trunk and a small dopaminergic innervation of the sensory cell areas. Surgical cuts in the brainstem confirm these dopaminergic fibers as part of the olivocochlear efferent bundle. Within the sensory epithelium, TH-positive terminals are seen only in the inner hair cell area, where they intermingle with other olivocochlear terminals expressing cholinergic markers (vesicular acetylcholine transporter; VAT). Double immunostaining suggests little colocalization of TH and VAT; quantification of terminal volumes suggests that TH-positive fibers constitute only 10-20% of the efferent innervation of the inner hair cell area. Immunostaining of mouse brainstem revealed a small population of TH-positive cells in and around the lateral superior olive. Consistent with cochlear projections, double staining for the cholinergic marker acetylcholinesterase suggested that TH-positive somata are not cholinergic and vice versa. All observations are consistent with the view that a small dopaminergic subgroup of lateral olivocochlear neurons 1) projects to the inner hair cell area, 2) is distinct from the larger cholinergic group projecting there, and 3) may correspond to lateral olivocochlear "shell" neurons described by others (Warr et al. [1997] Hear. Res 108:89-111).