The effect of progressive hearing loss on the morphology of endbulbs of Held and bushy cells.

Studies of congenital and early-onset deafness have demonstrated that an absence of peripheral sound-evoked activity in the auditory nerve causes pathological changes in central auditory structures. The aim of this study was to establish whether progressive acquired hearing loss could lead to similar brain changes that would degrade the precision of signal transmission. We used complementary physiologic hearing tests and microscopic techniques to study the combined effect of both magnitude and duration of hearing loss on one of the first auditory synapses in the brain, the endbulb of Held (EB), along with its bushy cell (BC) target in the anteroventral cochlear nucleus. We compared two hearing mouse strains (CBA/Ca and heterozygous shaker-2+/-) against a model of early-onset progressive hearing loss (DBA/2) and a model of congenital deafness (homozygous shaker-2-/-), examining each strain at 1, 3, and 6 months of age. Furthermore, we employed a frequency model of the mouse cochlear nucleus to constrain our analyses to regions most likely to exhibit graded changes in hearing function with time. No significant differences in the gross morphology of EB or BC structure were observed in 1-month-old animals, indicating uninterrupted development. However, in animals with hearing loss, both EBs and BCs exhibited a graded reduction in size that paralleled the hearing loss, with the most severe pathology seen in deaf 6-month-old shaker-2-/- mice. Ultrastructural pathologies associated with hearing loss were less dramatic: minor changes were observed in terminal size but mitochondrial fraction and postsynaptic densities remained relatively stable. These results indicate that acquired progressive hearing loss can have consequences on auditory brain structure, with prolonged loss leading to greater pathologies. Our findings suggest a role for early intervention with assistive devices in order to mitigate long-term pathology and loss of function.

Salicylate-Induced Hearing Loss Trigger Structural Synaptic Modifications in the Ventral Cochlear Nucleus of Rats via Medial Olivocochlear (MOC) Feedback Circuit.

Lesion-induced cochlear damage can result in synaptic outgrowth in the ventral cochlear nucleus (VCN). Tinnitus may be associated with the synaptic outgrowth and hyperactivity in the VCN. However, it remains unclear how hearing loss triggers structural synaptic modifications in the VCN of rats subjected to salicylate-induced tinnitus. To address this issue, we evaluated tinnitus-like behavior in rats after salicylate treatment and compared the amplitude of the distortion product evoked otoacoustic emission (DPOAE) and auditory brainstem response (ABR) between control and treated rats. Moreover, we observed the changes in the synaptic ultrastructure and in the expression levels of growth-associated protein (GAP-43), brain-derived neurotrophic factor (BDNF), the microglial marker Iba-1 and glial fibrillary acidic protein (GFAP) in the VCN. After salicylate treatment (300 mg/kg/day for 4 and 8 days), analysis of the gap prepulse inhibition of the acoustic startle showed that the rats were experiencing tinnitus. The changes in the DPOAE and ABR amplitude indicated an improvement in cochlear sensitivity and a reduction in auditory input following salicylate treatment. The treated rats displayed more synaptic vesicles and longer postsynaptic density in the VCN than the control rats. We observed that the GAP-43 expression, predominantly from medial olivocochlear (MOC) neurons, was significantly up-regulated, and that BDNF- and Iba-1-immunoreactive cells were persistently decreased after salicylate administration. Furthermore, GFAP-immunoreactive astrocytes, which is associated with synaptic regrowth, was significantly increased in the treated groups. Our study revealed that reduced auditory nerve activity triggers synaptic outgrowth and hyperactivity in the VCN via a MOC neural feedback circuit. Structural synaptic modifications may be a reflexive process that compensates for the reduced auditory input after salicylate administration. However, massive increases in excitatory synapses in the VCN may represent a detrimental process that causes central hyperactivity, leading to tinnitus.

Reversible neurotoxicity of kanamycin on dorsal cochlear nucleus.

The time course of aminoglycoside neurotoxic effect on cochlear nucleus is still obscure. We examined dynamic pathological changes of dorsal cochlear nucleus (DCN) and investigated whether apoptosis or autophagy was upregulated in the neurotoxic course of kanamycin on DCN after kanamycin treatment. Rats were treated with kanamycin sulfate/kg/day at a dose of 500mg by subcutaneous injection for 10 days. Dynamic pathological changes, neuron density and neuron apoptosis of the DCN were examined at 1, 7, 14, 28, 56, 70 and 140 days after kanamycin treatment. The expressions of JNK1, DAPK2, Bcl-2, p-Bcl-2, Caspase-3, LC3B and Beclin-1 were also detected. Under transmission electron microscopy, the mitochondrial swelling and focal vacuoles as well as endoplasmic reticulum dilation were progressively aggravated from 1 day to 14 days, and gradually recovered from 28 days to 140 days. Meanwhile, both autophagosomes and autolysosomes were increased from 1 day to 56 days. Only few neurons were positive to the TUNEL staining. Moreover, neither the expressions of caspase-3 and DAPK2 nor neurons density of DCN changed significantly. LC3-II was drastically increased at 7 days. Beclin-1 was upgraded at 1 and 7 days. P-Bcl-2 increased at 1, 7, 14 and 28 days. JNK1 increased at 7 days, and Bcl-2 was downgraded at 140 days. LC3-B positive neurons were increased at 1, 7 and 14 days. These data demonstrated that the neurons damage of the DCN caused by kanamycin was reversible and autophagy was upregulated in the neurotoxic course of kanamycin on DCN through JNK1-mediated phosphorylation of Bcl-2 pathway.

Cochlear nucleus neurons redistribute synaptic AMPA and glycine receptors in response to monaural conductive hearing loss.

Neurons restore their function in response to external or internal perturbations and maintain neuronal or network stability through a homeostatic scaling mechanism. Homeostatic responses at synapses along the auditory system would be important for adaptation to normal and abnormal fluctuations in the sensory environment. We investigated at the electron microscopic level and after postembedding immunogold labeling whether projection neurons in the cochlear nucleus responded to modifications of auditory nerve activity. After unilaterally reducing the level of auditory inputs by approximately 20 dB by monaural earplugging, auditory nerve synapses on bushy cells somata and basal dendrites of fusiform cells of the ventral and dorsal cochlear nucleus, respectively, upregulated GluR3 AMPA receptor subunit, while inhibitory synapses decreased the expression of GlyRalpha1 subunit. These changes in expression levels were fully reversible once the earplug was removed, indicating that activity affects the trafficking of receptors at synapses. Excitatory synapses on apical dendrites of fusiform cells (parallel fibers) with different synaptic AMPA receptor subunit composition, were not affected by sound attenuation, as the expression levels of AMPA receptor subunits were the same as in normal hearing littermates. GlyRalpha1 subunit expression at inhibitory synapses on apical dendrites of fusiform cells was also found unaffected. Furthermore, fusiform and bushy cells of the contralateral side to the earplugging upregulated the GluR3 subunit at auditory nerve synapses. These results show that cochlear nucleus neurons innervated by the auditory nerve, are able to respond to small changes in sound levels by redistributing specific AMPA and glycine receptor subunits.

Projections of the second cervical dorsal root ganglion to the cochlear nucleus in rats.

Physiological, anatomical, and clinical data have demonstrated interactions between somatosensory and auditory brainstem structures. Spinal nerve projections influence auditory responses, although the nature of the pathway(s) is not known. To address this issue, we injected biotinylated dextran amine into the cochlear nucleus or dorsal root ganglion (DRG) at the second cervical segment (C2). Cochlear nucleus injections retrogradely labeled small ganglion cells in C2 DRG. C2 DRG injections produced anterograde labeling in the external cuneate nucleus, cuneate nucleus, nucleus X, central cervical nucleus, dorsal horn of upper cervical spinal segments, and cochlear nucleus. The terminal field in the cochlear nucleus was concentrated in the subpeduncular corner and lamina of the granule cell domain, where endings of various size and shapes appeared. Examination under an electron microscope revealed that the C2 DRG terminals contained numerous round synaptic vesicles and formed asymmetric synapses, implying depolarizing influences on the target cell. Labeled endings synapsed with the stalk of the primary dendrite of unipolar brush cells, distal dendrites of presumptive granule cells, and endings containing pleomorphic synaptic vesicles. These primary somatosensory projections contribute to circuits that are hypothesized to mediate integrative functions of hearing.

Restoration of auditory nerve synapses in cats by cochlear implants.

Congenital deafness results in abnormal synaptic structure in endings of the auditory nerve. If these abnormalities persist after restoration of auditory nerve activity by a cochlear implant, the processing of time-varying signals such as speech would likely be impaired. We stimulated congenitally deaf cats for 3 months with a six-channel cochlear implant. The device used human speech-processing programs, and cats responded to environmental sounds. Auditory nerve fibers exhibited a recovery of normal synaptic structure in these cats. This rescue of synapses is attributed to a return of spike activity in the auditory nerve and may help explain cochlear implant benefits in childhood deafness.

Acoustic stria: anatomy of physiologically characterized cells and their axonal projection patterns.

The mammalian cochlear nucleus (CN) has been a model structure to study the relationship between physiological and morphological cell classes. Several issues remain, in particular with regard to the projection patterns and physiology of neurons that exit the CN dorsally via the dorsal (DAS), intermediate (IAS), and commissural stria. We studied these neurons physiologically and anatomically using the intra-axonal labeling method. Multipolar cells with onset chopper (O(C)) responses innervated the ipsilateral ventral and dorsal CN before exiting the CN via the commissural stria. Upon reaching the midline they turned caudally to innervate the opposite CN. No collaterals were seen innervating any olivary complex nuclei. Octopus cells typically showed onset responses with little or no sustained activity. The main axon used the IAS and followed one of two routes occasionally giving off olivary complex collaterals on their way to the contralateral ventral nucleus of the lateral lemniscus (VNLL). Here they can have elaborate terminal arbors that surround VNLL cells. Fusiform and giant cells have overlapping but not identical physiology. Fusiform but not giant cells typically show pauser or buildup responses. Axons of both cells exit via the DAS and take the same course to reach the contralateral IC without giving off any collaterals en route.

Direct innervation of identified tectothalamic neurons in the inferior colliculus by axons from the cochlear nucleus.

The present study sought to identify tectothalamic neurons in the rat inferior colliculus that receive their innervation directly from the cochlear nuclei and to identify the axons that provide the innervation. A direct projection would bypass the binaural interactions of the superior olivary complex and provide the quickest route to the neocortex. Axons, primarily from the dorsal cochlear nucleus, were labeled with anterograde transport of dextran and terminated in the central nucleus of the inferior colliculus in a laminar pattern. Most labeled axons were thin and simply branched. Other axons were thicker, gnarly, less frequently observed and probably originated from the ventral cochlear nucleus. None had concentrated endbulbs or a nest of endings. Both types of axons terminated primarily in the central nucleus and layer 3 of the external cortex. This pattern suggests that the combination of these subdivisions in the rat are equivalent to the central nucleus as defined in other species. Tectothalamic neurons in the inferior colliculus in the same animals were identified by retrograde transport from the medial geniculate body and intracellular injection of Lucifer Yellow. A number of different cell types act as tectothalamic neurons and receive contacts from cochlear nucleus axons. These include flat cells (disc-shaped), less-flat cells and stellate cells. Two innervation patterns were seen: a combination of axosomatic and axodendritic contacts, and predominantly axodendritic contacts. Both patterns were seen in the central nucleus, but axosomatic contacts were seen less often in the other subdivisions. This is the first study to show direct connections between cochlear nuclear axons and identified tectothalamic neurons. The layers of axons from cochlear nuclei may provide convergent inputs to neurons in the inferior colliculus rather than the heavy inputs from single axons typical of lower auditory nuclei. Excitatory synapses made by axons from the cochlear nuclei on tectothalamic neurons may provide a substrate for rapid transmission of monaural information to the medial geniculate body.

Inputs to a physiologically characterized region of the inferior colliculus of the young adult CBA mouse.

Presbycusis is a sensory perceptual disorder involving loss of high-pitch hearing and reduced ability to process biologically relevant acoustic signals in noisy environments. The present investigation is part of an ongoing series of studies aimed at discerning the neural bases of presbycusis. The purpose of the present experiment was to delineate the inputs to a functionally characterized region of the dorsomedial inferior colliculus (IC, auditory midbrain) in young, adult CBA mice. Focal, iontophoretic injections of horseradish peroxidase were made in the 18-24 kHz region of dorsomedial IC of the CBA strain following physiological mapping experiments. Serial sections were reacted with diaminobenzidine or tetramethylbenzidine, counterstained and examined for retrogradely labeled cell bodies. Input projections were observed contralaterally from: all three divisions of cochlear nucleus; intermediate and dorsal nuclei of the lateral lemniscus (LL); and the central nucleus, external nucleus and dorsal cortex of the IC. Input projections were observed ipsilaterally from: the medial and lateral superior olivary nuclei; the superior paraolivary nucleus; the dorsolateral and anterolateral periolivary nuclei; the dorsal and ventral divisions of the ventral nucleus of LL; the dorsal and intermediate nuclei of LL; the central nucleus, external nucleus and dorsal cortex of the IC outside the injection site; and small projections from central gray and the medial geniculate body. These findings in young, adult mice with normal hearing can now serve as a baseline for similar experiments being conducted in mice of older ages and with varying degrees of hearing loss to discover neural changes that may cause age-related hearing disorders.

Kangaroo rats exhibit spongiform degeneration of the central auditory system similar to that found in gerbils.

Kangaroo rats develop spongiform degeneration of the central auditory system similar to that seen in the gerbil. Light microscopic and transmission electron microscopic study of the cochlear nucleus and auditory nerve root (ANR) of Dipodomys deserti and D. merriami show that spongiform lesions develop in dendrites and oligodendrocytes of the cochlear nucleus and in oligodendrocytes of the ANR that are morphologically indistinguishable from those extensively described in the Mongolian gerbil, Meriones unguiculatus. As in Mongolian gerbils, the spongiform degeneration in Dipodomys were much more numerous in animals continually exposed to modest levels of low-frequency noise (< 75 dB SPL). The kangaroo rats with extensive spongiform degeneration also show slightly, but significantly, elevated auditory brainstem evoked response (ABR) thresholds to low-frequency stimuli, a result also found in Mongolian gerbils. These results suggest that the elevated ABR thresholds may be the result of spongiform degeneration. Because low-frequency noise-induced spongiform degeneration has now been shown in the cochlear nucleus of animals from separate families of Rodentia (Heteromyidae and Muridae), the possibility should be investigated that similar noise-induced degenerative changes occur in the central auditory system of other mammals with good low-frequency hearing.

Stimulus parameters affecting tissue injury during microstimulation in the cochlear nucleus of the cat.

We investigated the effects of continuous microstimulation in the cats' posteroventral cochlear nucleus, using chronically implanted activated iridium microelectrodes. We examined 51 electrode sites (39 pulsed sites, and 12 unpulsed sites). Seven hours of continuous stimulation at 500 Hz often produced tissue injury near the tips of the pulsed microelectrodes. The damage took the form of a region of vacuolated tissue extending 200 microns or more from the site of the electrode tip. Electron microscope studies showed the vacuoles to be severely edematous segments of myelinated axons. The statistical correlation between the amount of damaged tissue and the charge per phase was large and highly significant (P < 0.0001). When the electrodes were pulsed for 7 h at 500 Hz with charge-balanced biphasic pulse pairs, the threshold for the damage was approximately 3 nC/phase. The damage threshold was not appreciably lower than the stimulation protocol was extended to 35 h (7 h/day for 5 days). In contrast, the threshold for exciting neurons near the microelectrode is approximately 1 nC/phase, as determined by the evoked response recorded in the inferior colliculus. There was little correlation between the severity of the tissue damage and the geometric charge density at the surface of the electrodes, between the damage and amplitude of the cathodic phase of the voltage transient induced across the stimulating electrodes by the stimulus current pulses, or between the damage and the stimulus pulse duration.

Auditory nerve terminals in the cochlear nucleus magnocellularis: differences between low and high frequencies.

Primary auditory nerve fibers were labelled in the barn owl by localized horseradish peroxidase (HRP) injections into the cochlear nucleus angularis. They were followed to their terminal sites in the hearing organ (basilar papilla), confirming that they were auditory, and to the cochlear nucleus magnocellularis. The terminal sites of low-frequency fibers within nucleus magnocellularis always included an area previously described as the lagenar part, i.e., an area receiving primary input which is probably only vestibular. Furthermore, a number of differences were recognized between these low-frequency (up to 0.64 kHz) and the high-frequency (1.8 kHz and above) auditory nerve projections to nucleus magnocellularis. Most importantly, the collaterals given off by low-frequency fibers into the nucleus typically showed multiple terminal branching, with both en passant and terminal bouton-like swellings. High-frequency fiber collaterals, in contrast, terminated unbranched in a single endbulb of Held. Nucleus magnocellularis is the first station in a brainstem auditory pathway processing stimulus timing information, coded through neuronal phase locking. The prominent difference in terminal shape found between its low- and high-frequency input fibers is interpreted as reflecting different requirements of the absolute temporal precision for significant phase locking. Terminals in the shape of endbulbs of Held are probably a specialization to improve the temporal precision of synaptic transmission, allowing phase locking to higher frequencies.