Neurotrophin gene therapy to promote survival of spiral ganglion neurons after deafness.

Hearing impairment is a major health and economic concern worldwide. Currently, the cochlear implant (CI) is the standard of care for remediation of severe to profound hearing loss, and in general, contemporary CIs are highly successful. But there is great variability in outcomes among individuals, especially in children, with many CI users deriving much less or even marginal benefit. Much of this variability is related to differences in auditory nerve survival, and there has been substantial interest in recent years in exploring potential therapies to improve survival of the cochlear spiral ganglion neurons (SGN) after deafness. Preclinical studies using osmotic pumps and other approaches in deafened animal models to deliver neurotrophic factors (NTs) directly to the cochlea have shown promising results, especially with Brain-Derived Neurotrophic Factor (BDNF). More recent studies have focused on the use of NT gene therapy to force expression of NTs by target cells within the cochlea. This could provide the means for a one-time treatment to promote long-term NT expression and improve neural survival after deafness. This review summarizes the evidence for the efficacy of exogenous NTs in preventing SGN degeneration after hearing loss and reviews the animal research to date suggesting that NT gene therapy can elicit long-term NT expression in the cochlea, resulting in significantly improved SGN and radial nerve fiber survival after deafness. In addition, we discuss NT gene therapy in other non-auditory applications and consider some of the remaining issues with regard to selecting optimal vectors, timing of treatment, and place/method of delivery, etc. that must be resolved prior to considering clinical application.

AAV-Mediated Neurotrophin Gene Therapy Promotes Improved Survival of Cochlear Spiral Ganglion Neurons in Neonatally Deafened Cats: Comparison of AAV2-hBDNF and AAV5-hGDNF.

Outcomes with contemporary cochlear implants (CI) depend partly upon the survival and condition of the cochlear spiral ganglion (SG) neurons. Previous studies indicate that CI stimulation can ameliorate SG neural degeneration after deafness, and brain-derived neurotrophic factor (BDNF) delivered by an osmotic pump can further improve neural survival. However, direct infusion of BDNF elicits undesirable side effects, and osmotic pumps are impractical for clinical application. In this study, we explored the potential for two adeno-associated viral vectors (AAV) to elicit targeted neurotrophic factor expression in the cochlea and promote improved SG and radial nerve fiber survival. Juvenile cats were deafened prior to hearing onset by systemic aminoglycoside injections. Auditory brainstem responses showed profound hearing loss by 16-18 days postnatal. At ~ 4 weeks of age, AAV2-GFP (green fluorescent protein), AAV5-GFP, AAV2-hBDNF, or AAV5-hGDNF (glial-derived neurotrophic factor) was injected through the round window unilaterally. For GFP immunofluorescence, animals were studied ~ 4 weeks post-injection to assess cell types transfected and their distributions. AAV2-GFP immunofluorescence demonstrated strong expression of the GFP reporter gene in residual inner (IHCs), outer hair cells (OHCs), inner pillar cells, and in some SG neurons throughout the cochlea. AAV5-GFP elicited robust transduction of IHCs and some SG neurons, but few OHCs and supporting cells. After AAV-neurotrophic factor injections, animals were studied ~ 3 months post-injection to evaluate neural survival. AAV5-hGDNF elicited a modest neurotrophic effect, with 6 % higher SG density, but had no trophic effect on radial nerve fiber survival, and undesirable ectopic fiber sprouting occurred. AAV2-hBDNF elicited a similar 6 % increase in SG survival, but also resulted in greatly improved radial nerve fiber survival, with no ectopic fiber sprouting. A further study assessed whether AAV2-hBDNF neurotrophic effects would persist over longer post-injection periods. Animals examined 6 months after virus injection showed substantial neurotrophic effects, with 14 % higher SG density and greatly improved radial nerve fiber survival. Our results suggest that AAV-neurotrophin gene therapy can elicit expression of physiological concentrations of neurotrophins in the cochlea, supporting improved SG neuronal and radial nerve fiber survival while avoiding undesirable side effects. These studies also demonstrate the potential for application of cochlear gene therapy in a large mammalian cochlea comparable to the human cochlea and in an animal model of congenital/early acquired deafness.

Virally Mediated Overexpression of Glial-Derived Neurotrophic Factor Elicits Age- and Dose-Dependent Neuronal Toxicity and Hearing Loss.

Contemporary cochlear implants (CI) are generally very effective for remediation of severe to profound sensorineural hearing loss, but outcomes are still highly variable. Auditory nerve survival is likely one of the major factors underlying this variability. Neurotrophin therapy therefore has been proposed for CI recipients, with the goal of improving outcomes by promoting improved survival of cochlear spiral ganglion neurons (SGN) and/or residual hair cells. Previous studies have shown that glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor, and neurotrophin-3 can rescue SGNs following insult. The current study was designed to determine whether adeno-associated virus vector serotype 5 (AAV-5) encoding either green fluorescent protein or GDNF can transduce cells in the mouse cochlea to express useful levels of neurotrophin and to approximate the optimum therapeutic dose(s) for transducing hair cells and SGN. The findings demonstrate that AAV-5 is a potentially useful gene therapy vector for the cochlea, resulting in extremely high levels of transgene expression in the cochlear inner hair cells and SGN. However, overexpression of human GDNF in newborn mice caused severe neurological symptoms and hearing loss, likely due to Purkinje cell loss and cochlear nucleus pathology. Thus, extremely high levels of transgene protein expression should be avoided, particularly for proteins that have neurological function in neonatal subjects.

Passive stimulation and behavioral training differentially transform temporal processing in the inferior colliculus and primary auditory cortex.

In profoundly deaf cats, behavioral training with intracochlear electric stimulation (ICES) can improve temporal processing in the primary auditory cortex (AI). To investigate whether similar effects are manifest in the auditory midbrain, ICES was initiated in neonatally deafened cats either during development after short durations of deafness (8 wk of age) or in adulthood after long durations of deafness (≥3.5 yr). All of these animals received behaviorally meaningless, "passive" ICES. Some animals also received behavioral training with ICES. Two long-deaf cats received no ICES prior to acute electrophysiological recording. After several months of passive ICES and behavioral training, animals were anesthetized, and neuronal responses to pulse trains of increasing rates were recorded in the central (ICC) and external (ICX) nuclei of the inferior colliculus. Neuronal temporal response patterns (repetition rate coding, minimum latencies, response precision) were compared with results from recordings made in the AI of the same animals (Beitel RE, Vollmer M, Raggio MW, Schreiner CE. J Neurophysiol 106: 944-959, 2011; Vollmer M, Beitel RE. J Neurophysiol 106: 2423-2436, 2011). Passive ICES in long-deaf cats remediated severely degraded temporal processing in the ICC and had no effects in the ICX. In contrast to observations in the AI, behaviorally relevant ICES had no effects on temporal processing in the ICC or ICX, with the single exception of shorter latencies in the ICC in short-deaf cats. The results suggest that independent of deafness duration passive stimulation and behavioral training differentially transform temporal processing in auditory midbrain and cortex, and primary auditory cortex emerges as a pivotal site for behaviorally driven neuronal temporal plasticity in the deaf cat. NEW & NOTEWORTHY: Behaviorally relevant vs. passive electric stimulation of the auditory nerve differentially affects neuronal temporal processing in the central nucleus of the inferior colliculus (ICC) and the primary auditory cortex (AI) in profoundly short-deaf and long-deaf cats. Temporal plasticity in the ICC depends on a critical amount of electric stimulation, independent of its behavioral relevance. In contrast, the AI emerges as a pivotal site for behaviorally driven neuronal temporal plasticity in the deaf auditory system.

Effects of brain-derived neurotrophic factor (BDNF) on the cochlear nucleus in cats deafened as neonates.

Many previous studies have shown significant neurotrophic effects of intracochlear delivery of BDNF in preventing degeneration of cochlear spiral ganglion (SG) neurons after deafness in rodents and our laboratory has shown similar results in developing cats deafened prior to hearing onset. This study examined the morphology of the cochlear nucleus (CN) in a group of neonatally deafened cats from a previous study in which infusion of BDNF elicited a significant improvement in survival of the SG neurons. Five cats were deafened by systemic injections of neomycin sulfate (60 mg/kg, SQ, SID) starting one day after birth, and continuing for 16-18 days until auditory brainstem response (ABR) testing demonstrated profound bilateral hearing loss. The animals were implanted unilaterally at about 1 month of age using custom-designed electrodes with a drug-delivery cannula connected to an osmotic pump. BDNF (94 µg/ml; 0.25 µl/hr) was delivered for 10 weeks. The animals were euthanized and studied at 14-23 weeks of age. Consistent with the neurotrophic effects of BDNF on SG survival, the total CN volume in these animals was significantly larger on the BDNF-treated side than on the contralateral side. However, total CN volume, both ipsi- and contralateral to the implants in these deafened juvenile animals, was markedly smaller than the CN in normal adult animals, reflecting the severe effects of deafness on the central auditory system during development. Data from the individual major CN subdivisions (DCN, Dorsal Cochlear Nucleus; PVCN, Posteroventral Cochlear Nucleus; AVCN, Anteroventral Cochlear Nucleus) also were analyzed. A significant difference was observed between the BDNF-treated and control sides only in the AVCN. Measurements of the cross-sectional areas of spherical cells showed that cells were significantly larger in the AVCN ipsilateral to the implant than on the contralateral side. Further, the numerical density of spherical cells was significantly lower in the AVCN ipsilateral to the implant than on the contralateral side, consistent with the larger AVCN volume observed with BDNF treatment. Together, findings indicate significant neurotrophic effects of intracochlear BDNF infusion on the developing CN.

Effects of brain-derived neurotrophic factor (BDNF) and electrical stimulation on survival and function of cochlear spiral ganglion neurons in deafened, developing cats.

Both neurotrophic support and neural activity are required for normal postnatal development and survival of cochlear spiral ganglion (SG) neurons. Previous studies in neonatally deafened cats demonstrated that electrical stimulation (ES) from a cochlear implant can promote improved SG survival but does not completely prevent progressive neural degeneration. Neurotrophic agents combined with an implant may further improve neural survival. Short-term studies in rodents have shown that brain-derived neurotrophic factor (BDNF) promotes SG survival after deafness and may be additive to trophic effects of stimulation. Our recent study in neonatally deafened cats provided the first evidence of BDNF neurotrophic effects in the developing auditory system over a prolonged duration Leake et al. (J Comp Neurol 519:1526-1545, 2011). Ten weeks of intracochlear BDNF infusion starting at 4 weeks of age elicited significant improvement in SG survival and larger soma size compared to contralateral. In the present study, the same deafening and BDNF infusion procedures were combined with several months of ES from an implant. After combined BDNF + ES, a highly significant increase in SG numerical density (>50 % improvement re: contralateral) was observed, which was significantly greater than the neurotrophic effect seen with ES-only over comparable durations. Combined BDNF + ES also resulted in a higher density of myelinated radial nerve fibers within the osseous spiral lamina. However, substantial ectopic and disorganized sprouting of these fibers into the scala tympani also occurred, which may be deleterious to implant function. EABR thresholds improved (re: initial thresholds at time of implantation) on the chronically stimulated channels of the implant. Terminal electrophysiological studies recording in the inferior colliculus (IC) revealed that the basic cochleotopic organization was intact in the midbrain in all studied groups. In deafened controls or after ES-only, lower IC thresholds were correlated with more selective activation widths as expected, but no such correlation was seen after BDNF + ES due to much greater variability in both measures.

Monopolar intracochlear pulse trains selectively activate the inferior colliculus.

Previous cochlear implant studies using isolated electrical stimulus pulses in animal models have reported that intracochlear monopolar stimulus configurations elicit broad extents of neuronal activation within the central auditory system-much broader than the activation patterns produced by bipolar electrode pairs or acoustic tones. However, psychophysical and speech reception studies that use sustained pulse trains do not show clear performance differences for monopolar versus bipolar configurations. To test whether monopolar intracochlear stimulation can produce selective activation of the inferior colliculus, we measured activation widths along the tonotopic axis of the inferior colliculus for acoustic tones and 1,000-pulse/s electrical pulse trains in guinea pigs and cats. Electrical pulse trains were presented using an array of 6-12 stimulating electrodes distributed longitudinally on a space-filling silicone carrier positioned in the scala tympani of the cochlea. We found that for monopolar, bipolar, and acoustic stimuli, activation widths were significantly narrower for sustained responses than for the transient response to the stimulus onset. Furthermore, monopolar and bipolar stimuli elicited similar activation widths when compared at stimulus levels that produced similar peak spike rates. Surprisingly, we found that in guinea pigs, monopolar and bipolar stimuli produced narrower sustained activation than 60 dB sound pressure level acoustic tones when compared at stimulus levels that produced similar peak spike rates. Therefore, we conclude that intracochlear electrical stimulation using monopolar pulse trains can produce activation patterns that are at least as selective as bipolar or acoustic stimulation.

Brain-derived neurotrophic factor promotes cochlear spiral ganglion cell survival and function in deafened, developing cats.

Postnatal development and survival of spiral ganglion (SG) neurons depend on both neural activity and neurotrophic support. Our previous studies showed that electrical stimulation from a cochlear implant only partially prevents SG degeneration after early deafness. Thus, neurotrophic agents that might be combined with an implant to improve neural survival are of interest. Recent studies reporting that brain-derived neurotrophic factor (BDNF) promotes SG survival after deafness have been conducted in rodents and limited to relatively short durations. Our study examined longer duration BDNF treatment in deafened cats that may better model the slow progression of SG degeneration in human cochleae, and this is the first study of BDNF in the developing auditory system. Kittens were deafened neonatally, implanted at 4-5 weeks with intracochlear electrodes containing a drug-delivery cannula, and BDNF or artificial perilymph was infused for 10 weeks from a miniosmotic pump. In BDNF-treated cochleae, SG cells grew to normal size and were significantly larger than cells on the contralateral side. However, their morphology was not completely normal, and many neurons lacked or had thinned perikaryl myelin. Unbiased stereology was employed to estimate SG cell density, independent of cell size. BDNF was effective in promoting significantly improved survival of SG neurons in these developing animals. BDNF treatment also resulted in higher density and larger size of myelinated radial nerve fibers, sprouting of fibers into the scala tympani, and improvement of electrically evoked auditory brainstem response thresholds. BDNF may have potential therapeutic value in the developing auditory system, but many serious obstacles currently preclude clinical application.

Consensus panel on a cochlear coordinate system applicable in histologic, physiologic, and radiologic studies of the human cochlea.

HYPOTHESIS: An objective cochlear framework, for evaluation of the cochlear anatomy and description of the position of an implanted cochlear implant electrode, would allow the direct comparison of measures performed within the various subdisciplines involved in cochlear implant research. BACKGROUND: Research on the human cochlear anatomy in relation to tonotopy and cochlear implantation is conducted by specialists from numerous disciplines such as histologists, surgeons, physicists, engineers, audiologists, and radiologists. To allow accurate comparisons between and combinations of previous and forthcoming scientific and clinical studies, cochlear structures and electrode positions must be specified in a consistent manner. METHODS: Researchers with backgrounds in the various fields of inner ear research as well as representatives of the different manufacturers of cochlear implants (Advanced Bionics Corp., Med-El, Cochlear Corp.) were involved in consensus meetings held in Dallas, March 2005, and Asilomar, August 2005. Existing coordinate systems were evaluated, and requisites for an objective cochlear framework were discussed. RESULTS: The consensus panel agreed upon a 3-dimensional, cylindrical coordinate system of the cochlea using the "Cochlear View" as a basis and choosing a z axis through the modiolus. The zero reference angle was chosen at the center of the round window, which has a close relationship to the basal end of the Organ of Corti. CONCLUSION: Consensus was reached on an objective cochlear framework, allowing the outcomes of studies from different fields of research to be compared directly.

Considerations for design of future cochlear implant electrode arrays: electrode array stiffness, size, and depth of insertion.

The level of hearing rehabilitation enjoyed by cochlear implant (CI) recipients has increased dramatically since the introduction of these devices. This improvement is the result of continual development of these systems and the inclusion of subjects with less severe auditory pathology. Developments include advanced signal processing, higher stimulation rates, greater numbers of channels, and more efficient electrode arrays that are less likely to produce insertion damage. New directions in the application of CIs, particularly in combined acoustic and electrical stimulation, and increasing performance expectations will place greater demands on future electrode arrays. Specifically, the next generation of arrays must be reliably inserted without damage, must maintain residual acoustic function, and may need to be inserted more deeply. In this study, we measured the mechanical properties of eight clinical and prototype human CI electrode arrays and evaluated insertion trauma and insertion depth in 79 implanted cadaver temporal bones. We found that the size and shape of the array directly affect the incidence of observed trauma. Further, arrays with greater stiffness in the plane perpendicular to the plane of the cochlear spiral are less likely to cause severe trauma than arrays with similar vertical and horizontal stiffness.

Effects of age at onset of deafness and electrical stimulation on the developing cochlear nucleus in cats.

This study examined the effects of deafness and intracochlear electrical stimulation on the anatomy of the cochlear nucleus (CN) after a brief period of normal auditory development early in life. Kittens were deafened by systemic ototoxic drug injections either as neonates or starting at postnatal day 30. Total CN volume, individual CN subdivision volumes, and cross-sectional areas of spherical cell somata in the anteroventral CN (AVCN) were compared in neonatally deafened and 30-day deafened groups at 8 weeks of age and in young adults after approximately 6 months of electrical stimulation initiated at 8 weeks of age. Both neonatal and early acquired hearing loss resulted in a reduction in CN volume as compared to normal hearing cats. Comparison of 8- and 32-week old groups indicated that the CN continued to grow in both deafened groups despite the absence of auditory input. Preserving normal auditory input for 30 days resulted in a significant increase in both total CN volume and cross-sectional areas of spherical cell somata, as compared to neonatally deafened animals. Restoring auditory input in these developing animals by unilateral intracochlear electrical stimulation did not elicit any difference in CN volume between the two sides, but resulted in 7% larger spherical cell size on the stimulated side. Overall, the brief period of normal auditory development and subsequent electrical stimulation maintained CN volume at 80% of normal and spherical cell size at 86% of normal ipsilateral to the implant as compared to 67% and 74%, respectively, in the neonatally deafened group.

Topography of auditory nerve projections to the cochlear nucleus in cats after neonatal deafness and electrical stimulation by a cochlear implant.

We previously reported that auditory nerve projections from the cochlear spiral ganglion (SG) to the cochlear nucleus (CN) exhibit clear cochleotopic organization in adult cats deafened as neonates before hearing onset. However, the topographic specificity of these CN projections in deafened animals is proportionately broader than normal (less precise relative to the CN frequency gradient). This study examined SG-to-CN projections in adult cats that were deafened as neonates and received a unilateral cochlear implant at approximately 7 weeks of age. Following several months of electrical stimulation, SG projections from the stimulated cochleae were compared to projections from contralateral, non-implanted ears. The fundamental organization of SG projections into frequency band laminae was clearly evident, and discrete projections were always observed following double SG injections in deafened cochleae, despite severe auditory deprivation and/or broad electrical activation of the SG. However, when normalized for the smaller CN size after deafness, AVCN, PVCN, and DCN projections on the stimulated side were broader by 32%, 34%, and 53%, respectively, than projections in normal animals (although absolute projection widths were comparable to normal). Further, there was no significant difference between projections from stimulated and contralateral non-implanted cochleae. These findings suggest that early normal auditory experience may be essential for normal development and/or maintenance of the topographic precision of SG-to-CN projections. After early deafness, the CN is smaller than normal, the topographic distribution of these neural projections that underlie frequency resolution in the central auditory system is proportionately broader, and projections from adjacent SG sectors are more overlapping. Several months of stimulation by a cochlear implant (beginning at approximately 7 weeks of age) did not lessen or exacerbate these degenerative changes observed in adulthood. One clinical implication of these findings is that congenitally deaf cochlear implant recipients may have central auditory system alterations that limit their ability to achieve spectral selectivity equivalent to post-lingually deafened subjects.

Factors influencing neurotrophic effects of electrical stimulation in the deafened developing auditory system.

Research in animal models has demonstrated that electrical stimulation from a cochlear implant (CI) may help prevent degeneration of the cochlear spiral ganglion (SG) neurons after deafness. In cats deafened early in life, effective stimulation of the auditory nerve with complex signals for several months preserves a greater density of SG neurons in the stimulated cochleae as compared to the contralateral deafened ear. However, SG survival is still far from normal even with early intervention with an implant. Thus, pharmacologic agents and neurotrophic factors that might be used in combination with an implant are of great interest. Exogenous administration of GM1 ganglioside significantly reduces SG degeneration in deafened animals studied at 7-8 weeks of age, but after several months of stimulation, GM1-treated animals show only modestly better preservation of SG density compared to age-matched non-treated animals. A significant factor influencing neurotrophic effects in animal models is insertion trauma, which results in significant regional SG degeneration. Thus, an important goal is to further improve human CI electrode designs and insertion techniques to minimize trauma. Another important issue for studies of neurotrophic effects in the developing auditory system is the potential role of critical periods. Studies examining animals deafened at 30 days of age (rather than at birth) have explored whether a brief initial period of normal auditory experience affects the vulnerability of the SG or cochlear nucleus (CN) to auditory deprivation. Interestingly, SG survival in animals deafened at 30-days was not significantly different from age-matched neonatally deafened animals, but significant differences were observed in the central auditory system. CN volume was significantly closer to normal in the animals deafened at 30 days as compared to neonatally deafened animals. However, no difference was observed between the stimulated and contralateral CN volumes in either deafened group. Measurements of AVCN spherical cell somata showed that after later onset of deafness in the 30-day deafened group, mean cell size was significantly closer to normal than in the neonatally deafened group. Further, electrical stimulation elicited a significant increase in spherical cell size in the CN ipsilateral to the implant as compared to the contralateral CN in both deafened groups. Neuronal tracer studies have examined the primary afferent projections from the SG to the CN in neonatally deafened cats. CN projections exhibit a clear cochleotopic organization despite severe auditory deprivation from birth. However, when normalized for the smaller CN size after deafness, projections were 30-50% broader than normal. After unilateral electrical stimulation there was no difference between projections from the stimulated and non-stimulated ears. These findings suggest that early normal auditory experience may be essential for the normal development (or subsequent maintenance) of the topographic precision of SG-to-CN projections. After early deafness, the CN volume is markedly smaller than normal, and the spatial precision of SG projections that underlie frequency resolution in the central auditory system is reduced. Electrical stimulation over several months did not reduce or exacerbate these degenerative changes. If similar principles pertain in the human auditory system, then findings in animal models suggest that the basic cochleotopic organization of neural projections in the central auditory system is probably intact even in congenitally deaf individuals. However, the reduced spatial resolution of the primary afferent projections in our studies suggests that there may be inherent limitations for CI stimulation in congenitally deaf subjects. Spatial (spectral) selectivity of stimulation delivered on adjacent CI channels may be poorer due to the greater overlap of SG central axons representing nearby frequencies. Such CI users may be more dependent upon temporal features of electrical stimuli, and it may be advantageous to enhance the salience of such cues, for example, by removing some electrodes from the processor "map" to reduce channel interaction.

Spatial selectivity to intracochlear electrical stimulation in the inferior colliculus is degraded after long-term deafness in cats.

In an animal model of electrical hearing in prelingually deaf adults, this study examined the effects of deafness duration on response thresholds and spatial selectivity (i.e., cochleotopic organization, spatial tuning and dynamic range) in the central auditory system to intracochlear electrical stimulation. Electrically evoked auditory brain stem response (EABR) thresholds and neural response thresholds in the external (ICX) and central (ICC) nuclei of the inferior colliculus were estimated in cats after varying durations of neonatally induced deafness: in animals deafened <1.5 yr (short-deafened unstimulated, SDU cats) with a mean spiral ganglion cell (SGC) density of approximately 45% of normal and in animals deafened >2.5 yr (long-deafened, LD cats) with severe cochlear pathology (mean SGC density <7% of normal). LD animals were subdivided into unstimulated cats and those that received chronic intracochlear electrical stimulation via a feline cochlear implant. Acutely deafened, implanted adult cats served as controls. Independent of their stimulation history, LD animals had significantly higher EABR and ICC thresholds than SDU and control animals. Moreover, the spread of electrical excitation was significantly broader and the dynamic range significantly reduced in LD animals. Despite the prolonged durations of deafness the fundamental cochleotopic organization was maintained in both the ICX and the ICC of LD animals. There was no difference between SDU and control cats in any of the response properties tested. These findings suggest that long-term auditory deprivation results in a significant and possibly irreversible degradation of response thresholds and spatial selectivity to intracochlear electrical stimulation in the auditory midbrain.

Spontaneous discharge patterns in cochlear spiral ganglion cells before the onset of hearing in cats.

Spontaneous neural activity has been recorded in the auditory nerve of cats as early as 2 days postnatal (P2), yet individual auditory neurons do not respond to ambient sound levels <90-100 dB SPL until about P10. Significant refinement of the central projections from the spiral ganglion to the cochlear nucleus occurs during this neonatal period. This refinement may be dependent on peripheral spontaneous discharge activity. We recorded from single spiral ganglion cells in kittens aged P3-P9. The spiral ganglion was accessed through the round window through the spiral lamina. A total of 112 ganglion cells were isolated for study in nine animals. Spike rates in neonates were very low, ranging from 0.06 to 56 spikes/s, with a mean of 3.09 +/- 8.24 spikes/s. Ganglion cells in neonatal kittens exhibited remarkable repetitive spontaneous bursting discharge patterns. The unusual patterns were evident in the large mean interval CV (CV(i) = 2.9 +/- 1.6) and burst index of 5.2 +/- 3.5 across ganglion cells. Spontaneous bursting patterns in these neonatal mammals were similar to those reported for cochlear ganglion cells of the embryonic chicken, suggesting this may be a general phenomenon that is common across animal classes. Rhythmic spontaneous discharge of retinal ganglion cells has been shown to be important in the development of central retinotopic projections and normal binocular vision. Bursting rhythms in cochlear ganglion cells may play a similar role in the auditory system during prehearing periods.

Design and fabrication of multichannel cochlear implants for animal research.

The effectiveness of multichannel cochlear implants depends on the activation of perceptually distinct regions of the auditory nerve. Increased information transfer is possible as the number of channels and dynamic range are increased and electrical and neural interaction among channels is reduced. Human and animal studies have demonstrated that specific design features of the intracochlear electrode directly affect these performance factors. These features include the geometry, size, and orientation of the stimulating sites, proximity of the device to spiral ganglion neurons, shape and position of the insulating carrier, and the stimulation mode (monopolar, bipolar, etc.). Animal studies to directly measure the effects of changes in electrode design are currently constrained by the lack of available electrodes that model contemporary clinical devices. This report presents methods to design and fabricate species-specific customizable electrode arrays. We have successfully implanted these arrays in guinea pigs and cats for periods of up to 14 months and have conducted acute electrophysiological experiments in these animals. Modifications enabling long-term intracochlear drug infusion are also described. Studies using these scale model arrays will improve our understanding of how these devices function in human subjects and how we can best optimize future cochlear implants.

Neurotrophic effects of GM1 ganglioside and electrical stimulation on cochlear spiral ganglion neurons in cats deafened as neonates.

Previous studies have shown that electrical stimulation of the cochlea by a cochlear implant promotes increased survival of spiral ganglion (SG) neurons in animals deafened early in life (Leake et al. [1999] J Comp Neurol 412:543-562). However, electrical stimulation only partially prevents SG degeneration after deafening and other neurotrophic agents that may be used along with an implant are of great interest. GM1 ganglioside is a glycosphingolipid that has been reported to be beneficial in treating stroke, spinal cord injuries, and Alzheimer's disease. GM1 activates trkB signaling and potentiates neurotrophins, and exogenous administration of GM1 has been shown to reduce SG degeneration after hearing loss. In the present study, animals were deafened as neonates and received daily injections of GM1, beginning either at birth or after animals were deafened and continuing until the time of cochlear implantation. GM1-treated and deafened control groups were examined at 7-8 weeks of age; additional GM1 and no-GM1 deafened control groups received a cochlear implant at 7-8 weeks of age and at least 6 months of unilateral electrical stimulation. Electrical stimulation elicited a significant trophic effect in both the GM1 group and the no-GM1 group as compared to the contralateral, nonstimulated ears. The results also demonstrated a modest initial improvement in SG density with GM1 treatment, which was maintained by and additive with the trophic effect of subsequent electrical stimulation. However, in the deafened ears contralateral to the implant SG soma size was severely reduced several months after withdrawal of GM1 in the absence of electrical activation.

Frequency map for the human cochlear spiral ganglion: implications for cochlear implants.

The goals of this study were to derive a frequency-position function for the human cochlear spiral ganglion (SG) to correlate represented frequency along the organ of Corti (OC) to location along the SG, to determine the range of individual variability, and to calculate an "average" frequency map (based on the trajectories of the dendrites of the SG cells). For both OC and SG frequency maps, a potentially important limitation is that accurate estimates of cochlear place frequency based upon the Greenwood function require knowledge of the total OC or SG length, which cannot be determined in most temporal bone and imaging studies. Therefore, an additional goal of this study was to evaluate a simple metric, basal coil diameter that might be utilized to estimate OC and SG length. Cadaver cochleae (n = 9) were fixed <24 h postmortem, stained with osmium tetroxide, microdissected, decalcified briefly, embedded in epoxy resin, and examined in surface preparations. In digital images, the OC and SG were measured, and the radial nerve fiber trajectories were traced to define a series of frequency-matched coordinates along the two structures. Images of the cochlear turns were reconstructed and measurements of basal turn diameter were made and correlated with OC and SG measurements. The data obtained provide a mathematical function for relating represented frequency along the OC to that of the SG. Results showed that whereas the distance along the OC that corresponds to a critical bandwidth is assumed to be constant throughout the cochlea, estimated critical band distance in the SG varies significantly along the spiral. Additional findings suggest that measurements of basal coil diameter in preoperative images may allow prediction of OC/SG length and estimation of the insertion depth required to reach specific angles of rotation and frequencies. Results also indicate that OC and SG percentage length expressed as a function of rotation angle from the round window is fairly constant across subjects. The implications of these findings for the design and surgical insertion of cochlear implants are discussed.

A frequency-position function for the human cochlear spiral ganglion.

Greenwood's frequency-position function for the organ of Corti (OC) is commonly used to estimate represented frequencies for cochlear implant (CI) electrodes, both in temporal bone studies and in imaging studies of living CI recipients. However, many contemporary CIs position stimulating electrodes near the modiolus, directly targeting the spiral ganglion (SG) cells within Rosenthal's canal. At the extreme base and apex, the SG does not extend as far as the OC, and the radial nerve fibers take a tangential course into the modiolus resulting in a potential offset between the frequency maps of the OC and SG. In this investigation, human cadaveric cochleae (n = 7) were studied in surface preparations after osmium staining. The OC and SG lengths were measured and radial fiber trajectories traced to identify frequency-matched points on each structure. These data allowed derivation of a mathematical function correlating represented frequency along the OC to position along the SG. A cubic function fit the data with a very high intersubject correlation. Better knowledge of the human SG 'neural frequency map' may help to refine electrode design, and to more accurately map CI channel filter bands to the appropriate cochlear place along the SG, which may be advantageous for more sophisticated CI outcomes, such as music appreciation. These data also could be valuable for electroacoustic stimulation, by defining the insertion distance of a CI electrode required to reach specific frequencies (based upon preoperative imaging) in an individual subject, thus helping to avoid trauma to cochlear regions with residual hearing.

Neonatal deafness results in degraded topographic specificity of auditory nerve projections to the cochlear nucleus in cats.

We previously examined the early postnatal maturation of the primary afferent auditory nerve projections from the cat cochlear spiral ganglion (SG) to the cochlear nucleus (CN). In normal kittens these projections exhibit clear cochleotopic organization before birth, but quantitative data showed that their topographic specificity is less precise in perinatal kittens than in adults. Normalized for CN size, projections to the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) subdivisions are all significantly broader in neonates than in adults. By 6-7 postnatal days, projections are proportionate to those of adults, suggesting that significant refinement occurs during the early postnatal period. The present study examined SG projections to the CN in adult cats deafened as neonates by ototoxic drug administration. The fundamental organization of the SG-to-CN projections into frequency band laminae is clearly evident despite severe auditory deprivation from birth. However, when normalized for the smaller CN size in deafened animals, projections are disproportionately broader than in controls; AVCN, PVCN, and DCN projections are 39, 26, and 48% broader, respectively, than predicted if they were precisely proportionate to projections in normal hearing animals. These findings suggest that normal auditory experience and neural activity are essential for the early postnatal development (or subsequent maintenance) of the topographic precision of SG-to-CN projections. After early deafness, the basic cochleotopic organization of the CN is established and maintained into adulthood, but the CN is severely reduced in size and the topographic specificity of primary afferent projections that underlies frequency resolution in the normal central auditory system is significantly degraded.