Stem-cell therapy for hearing loss: are we there yet? / Terapia com células-tronco para perda auditiva: já chegamos lá?

Abstract Introduction: Mammalian hair cells and auditory neurons do not show regenerative capacity. Hence, damage to these cell types is permanent and leads to hearing loss. However, there is no treatment that re-establishes auditory function. Regenerative therapies using stem cells represent a promising alternative. Objective: This article aims to review the current literature about the main types of stem cells with potential for application in cell therapy for sensorineural hearing loss, the most relevant experiments already performed in animals, as well as the advances that have been recently made in the field. Methods: Research included the databases PubMed/MEDLINE, Web of Science, Science Direct and SciELO, as well as gray literature. Search strategy included the following main terms: "stem cells", "hair cells" and "auditory neurons". Additionally, the main terms were combined with the following secondary terms: "mesenchymal", "iPS", "inner ear", "auditory". The research was conducted independently by three researchers. Results: Differentiation of stem cells into hair cells and auditory neurons has a high success rate, reaching up to 82% for the first and 100% for the latter. Remarkably, these differentiated cells are able to interact with hair cells and auditory neurons of cochlear explants through formation of new synapses. When transplanted into the cochlea of animals with hearing loss, auditory restoration has been documented to date only in deafferented animals. Conclusion: Advances have been more prominent in cases of auditory neuropathy, since partial improvement of auditory nerve conditions through cell-based therapy may increase the number of patients who can successfully receive cochlear implants.

Resumo Introdução: Nos mamíferos, as células ciliadas e os neurônios auditivos não apresentam capacidade regenerativa. Assim, os danos a esses tipos celulares são permanentes e levam à perda auditiva. Contudo, como não há tratamento que restabeleça a função auditiva, as terapias regenerativas utilizando células-tronco representam uma alternativa promissora. Objetivo: Este artigo tem como objetivo revisar a literatura atual sobre os principais tipos de células-tronco com potencial para aplicação em terapia celular para perda auditiva sensorioneural, os experimentos mais relevantes já realizados em animais, bem como os avanços obtidos recentemente nessa área. Método: As pesquisas incluíram as bases de dados PubMed/MEDLINE, Web of Science, Science Direct e SciELO, além da literatura cinza. A estratégia de busca incluiu os seguintes termos principais: "stem cells", "hair cells" e "auditory neurons". Além disso, os termos principais foram combinados com os seguintes termos secundários: "mesenchymal", "iPS", "inner ear" e "auditory". A pesquisa foi realizada de forma independente por três pesquisadores. Resultados: A diferenciação de células-tronco em células ciliadas e neurônios auditivos têm alta taxa de sucesso, chegando a 82% para o primeiro caso e 100% para o segundo. Notavelmente, essas células diferenciadas são capazes de interagir com células ciliadas e neurônios auditivos de explantes cocleares através da formação de novas sinapses. Quando transplantadas para a cóclea de animais com perda auditiva, a restauração da função auditiva foi observada, até o momento, apenas em animais com ablação do VIII nervo craniano. Conclusão: Os avanços têm sido mais proeminentes em casos de neuropatia auditiva. A melhora parcial das condições do nervo auditivo por meio de terapia baseada em células-tronco pode aumentar o número de pacientes candidatos a receber implantes cocleares com sucesso.

Stem-cell therapy for hearing loss: are we there yet?

INTRODUCTION: Mammalian hair cells and auditory neurons do not show regenerative capacity. Hence, damage to these cell types is permanent and leads to hearing loss. However, there is no treatment that re-establishes auditory function. Regenerative therapies using stem cells represent a promising alternative. OBJECTIVE: This article aims to review the current literature about the main types of stem cells with potential for application in cell therapy for sensorineural hearing loss, the most relevant experiments already performed in animals, as well as the advances that have been recently made in the field. METHODS: Research included the databases PubMed/MEDLINE, Web of Science, Science Direct and SciELO, as well as gray literature. Search strategy included the following main terms: "stem cells", "hair cells" and "auditory neurons". Additionally, the main terms were combined with the following secondary terms: "mesenchymal", "iPS", "inner ear", "auditory". The research was conducted independently by three researchers. RESULTS: Differentiation of stem cells into hair cells and auditory neurons has a high success rate, reaching up to 82% for the first and 100% for the latter. Remarkably, these differentiated cells are able to interact with hair cells and auditory neurons of cochlear explants through formation of new synapses. When transplanted into the cochlea of animals with hearing loss, auditory restoration has been documented to date only in deafferented animals. CONCLUSION: Advances have been more prominent in cases of auditory neuropathy, since partial improvement of auditory nerve conditions through cell-based therapy may increase the number of patients who can successfully receive cochlear implants.

Temporal coupling between specifications of neuronal and macular fates of the inner ear.

The inner ear is a complex organ comprised of various specialized sensory organs for detecting sound and head movements. The timing of specification for these sensory organs, however, is not clear. Previous fate mapping results of the inner ear indicate that vestibular and auditory ganglia and two of the vestibular sensory organs, the utricular macula (UM) and saccular macula (SM), are lineage related. Based on the medial-lateral relationship where respective auditory and vestibular neuroblasts exit from the otic epithelium and the subsequent formation of the medial SM and lateral UM in these regions, we hypothesized that specification of the two lateral structures, the vestibular ganglion and the UM are coupled and likewise for the two medial structures, the auditory ganglion and the SM. We tested this hypothesis by surgically inverting the primary axes of the otic cup in ovo and investigating the fate of the vestibular neurogenic region, which had been spotted with a lipophilic dye. Our results showed that the laterally-positioned, dye-associated, vestibular ganglion and UM were largely normal in transplanted ears, whereas both auditory ganglion and SM showed abnormalities suggesting the lateral but not the medial-derived structures were mostly specified at the time of transplantation. Both of these results are consistent with a temporal coupling between neuronal and macular fate specifications.

Formation of the peripheral-central transitional zone in the postnatal mouse cochlear nerve.

OBJECTIVES: Determine the formation of peripheral and central nervous system (CNS and PNS) transitional zone (PCTZ) along the postnatal mouse cochlear nerve. STUDY DESIGN: Prospective, basic science. SETTING: Research laboratory. SUBJECTS AND METHODS: A novel cryosection model of cochlea-cochlear nerve-brainstem was used in this study. The sections were harvested from a total of 45 mice in 9 groups of postnatal-day-0 to postnatal-day-60 mice (n = 5). Differential interference contrast microscopy and immunofluorescence were used to study the formation of PCTZ along the cochlear nerve of the postnatal mouse. RESULTS: The CNS tissue extended peripherally along the cochlear nerve from postnatal-day-0 to postnatal-day-7 and then stably located at the level of the spiral lamina of the basal cochlear turn. The PCTZ reached a mature pattern along the cochlear nerve after postnatal-day-7. A long segment of the CNS tissue extended along the cochlear nerve in the postnatal mouse. CONCLUSION: In the early postnatal days, the PCTZ extended peripherally toward the cochlea and obtains a mature pattern along the neonatal mouse cochlear nerve.

Restoration of auditory evoked responses by human ES-cell-derived otic progenitors.

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells, is responsible for a substantial proportion of patients with hearing impairment. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Survival, migration and differentiation of mouse tau-GFP embryonic stem cells transplanted into the rat auditory nerve.

Stem cells have been investigated as treatment for a variety of diagnoses such as Parkinson's disease, Alzheimer's disease and spinal cord injuries. Here, we investigated the possibility of using stem cells as a replacement therapy for lesions of the auditory nerve (AN). We transplanted tau-GFP mouse embryonic stem cells into the AN either by the internal auditory meatus or via the modiolus in rats that had been previously deafened by application of ß-bungarotoxin to the round window niche. We investigated the effect of brain derived neurotrophic factor (BDNF) on cell transplant survival and differentiation. Additionally chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core chondroitin sulfate proteoglycans, was used in order to promote possible migration of cells and axons through the transitional zone. A bioactive isoleucine-lysine-valine-alanine-valine (IKVAV) peptide amphiphile (PA) nanofiber gel was applied around the cell injection site. This nanofiber gel has been shown to promote neural differentiation and other similar gels have been used to encapsulate and release proteins. Three weeks after injection, transplanted cells were found in the scala tympani, the modiolus, the AN trunk and the brain stem. As compared to cell transplantation and gel only, BDNF content in the PA gel increased cell survival and neuronal differentiation. In the animals treated with ChABC we observed extensive migration of cells through the transitional zone to or from the CNS.

The border between the central and the peripheral nervous system in the cat cochlear nerve: a light and scanning electron microscopical study.

The transition between the central (CNS) and peripheral nervous system (PNS) in cranial and spinal nerve roots, referred to here as the CNS-PNS border, is of relevance to nerve root disorders and factors that affect peripheral-central regeneration. Here, this border is described in the cat cochlear nerve using light microscopical sections, and scanning electron microscopy of the CNS-PNS interfaces exposed by fracture of the nerve either prior to or following critical point drying. The CNS-PNS border represents an abrupt change in type of myelin, supporting elements, and vascularization. Because central myelin is formed by oligodendrocytes and peripheral myelin by Schwann cells, the myelinated fibers are as a rule equipped with a node of Ranvier at the border passage. The border is shallower and smoother in cat cochlear nerve than expected from other nerves, and the borderline nodes are largely in register. The loose endoneurial connective tissue of the PNS compartment is closed at the border by a compact glial membrane, the mantle zone, of the CNS compartment. The mantle zone is penetrated by the nerve fibers, but is otherwise composed of astrocytes and their interwoven processes like the external limiting membrane of the brain surface with which it is continuous. The distal surface of the mantle zone is covered by a fenestrated basal lamina. Only occasional vessels traverse the border. From an anatomical point of view, the border might be expected to be a weak point along the cochlear nerve and thus vulnerable to trauma. In mature animals, the CNS-PNS border presents a barrier to regrowth of regenerating nerve fibers and to invasion of the CNS by Schwann cells. An understanding of this region in the cochlear nerve is therefore relevant to head injuries that lead to hearing loss, to surgery on acoustic Schwannomas, and to the possibility of cochlear nerve regeneration.

Histopathologic investigation of the dimensions of the cochlear nerve canal in normal temporal bones.

OBJECTIVE: Establish normative histopathologic data on the dimensions of the cochlear nerve canal (CNC). BACKGROUND: Evidence suggests that when the CNC is stenotic, the cochlear nerve may be hypoplastic. There is clear agreement in the literature that an internal auditory canal less than 2 mm in diameter is a relative contraindication to cochlear implantation in children. However, there has only been recent recognition in research that a narrowed CNC may lead to diminished ability to interpolate and use auditory information delivered through a cochlear implant. However, there is no consensus in the literature on the normal diameter of the CNC and what parameters should be used to determine stenosis. In addition, no normative histopathologic data is available for CNCs. METHODS: This study evaluated histopathologic axial sections from normal human temporal bones to measure the cochlear nerve canal in 110 individuals, aged 0-100 years. The maximum CNC diameter in each normal patient was identified and measured. RESULTS: The mean CNC diameter was 2.26 mm with a standard deviation of 0.25 mm. There were no differences in the CNC diameters between males and females or with increasing age. CONCLUSION: These measurements should provide a normative reference for comparison in histopathologic and radiographic assessment of any patient with suspected cochlear nerve canal stenosis.

MicroRNA-183 family members regulate sensorineural fates in the inner ear.

Members of the microRNA (miRNA) 183 family (miR-183, miR-96, and miR-182) are expressed abundantly in specific sensory cell types in the eye, nose, and inner ear. In the inner ear, expression is robust in the mechanosensory hair cells and weak in the associated statoacoustic ganglion (SAG) neurons; both cell types can share a common lineage during development. Recently, dominant-progressive hearing loss in humans and mice was linked to mutations in the seed region of miR-96, with associated defects in both development and maintenance of hair cells in the mutant mice. To understand how the entire triplet functions in the development of mechanosensory hair cells and neurons of the inner ear, we manipulated the levels of these miRNAs in zebrafish embryos using synthesized miRNAs and antisense morpholino oligonucleotides (MOs). Overexpression of miR-96 or miR-182 induces duplicated otocysts, ectopic or expanded sensory patches, and extra hair cells, whereas morphogenesis of the SAG is adversely affected to different degrees. In contrast, knockdown of miR-183, miR-96, and miR-182 causes reduced numbers of hair cells in the inner ear, smaller SAGs, defects in semicircular canals, and abnormal neuromasts on the posterior lateral line. However, the prosensory region of the posterior macula, where the number of hair cells is reduced by approximately 50%, is not significantly impaired. Our findings suggest both distinct and common roles for the three miRNAs in cell-fate determination in the inner ear, and these principles might apply to development of other sensory organs.

Over-expression of BDNF by adenovirus with concurrent electrical stimulation improves cochlear implant thresholds and survival of auditory neurons.

The survival of the auditory nerve in cases of sensorineural hearing loss is believed to be a major factor in effective cochlear implant function. The current study assesses two measures of cochlear implant thresholds following a post-deafening treatment intended to halt auditory nerve degeneration. We used an adenoviral construct containing a gene insert for brain-derived neurotrophic factor (BDNF), a construct that has previously been shown to promote neuronal survival in a number of biological systems. We implanted ototoxically deafened guinea pigs with a multichannel cochlear implant and delivered a single inoculation of an adenovirus suspension coding for BDNF (Ad.BDNF) into the scala tympani at the time of implantation. Thresholds to electrical stimulation were assessed both psychophysically and electrophysiologically over a period of 80 days. Spiral ganglion cell survival was analyzed at the 80 days time point. Compared to the control group, the Ad.BDNF treated group had lower psychophysical and electrophysiological thresholds as well as higher survival of spiral ganglion cells. Electrophysiological, but not psychophysical, thresholds correlated well with the density of spiral ganglion cells. These results indicate that the changes in the anatomy of the auditory nerve induced by the combination of Ad.BDNF inoculation and the electrical stimulation used for testing improved functional measures of CI performance.

Stem cell transplantation for auditory nerve replacement.

The successful function of cochlear prostheses depends on activation of auditory nerve. The survival of auditory nerve neurons, however, can vary widely in candidates for cochlear implants and influence implant efficacy. Stem cells offer the potential for improving the function of cochlear prostheses and increasing the candidate pool by replacing lost auditory nerve. The first phase of studies for stem cell replacement of auditory nerve has examined the in vitro survival and differentiation as well as in vivo differentiation and survival of exogenous embryonic and tissue stem cells placed into scala tympani and/or modiolus. These studies are reviewed and new results on in vivo placement of B-5 mouse embryonic stem cells into scala tympani of the guinea pig cochleae with differentiation into a glutamatergic neuronal phenotype are presented. Research on the integration and connections of stem cell derived neurons in the cochlea is described. Finally, an alternative approach is considered, based on the use of endogenous progenitors rather than exogenous stem cells, with a review of promising findings that have identified stem cell-like progenitors in cochlear and vestibular tissues to provide the potential for auditory nerve replacement.

A cell therapy approach to substitute neural elements in the inner ear.

Three different donor tissues were tested for their capacity to survive, integrate and differentiate in the adult inner ear. Surviving embryonic dorsal root ganglion cells were found within the spiral ganglion neuron region and along the auditory nerve fibers. In the presence of exogenous nerve growth factor (NGF), the dorsal root ganglion cells formed extensive growth of neurites that seemed to contact the host neurons. Adult neural stem cells survived relative poorly in the inner ear whereas embryonic stem cells showed a somewhat greater capacity for survival and integration. Overall, the survival rate of implanted tissue was quite low in the cochlea. It is concluded that an inner ear cell therapy approach based on the implantation of exogenous cells will require that important survival factors are identified and supplied. In addition, it is possible that the physical properties of the cochlea, e.g., fluid-filled compartments and very limited space for cell proliferation, are unfavorable, at least in the normal cochlea.

Migration of neural precursor cells derived from olfactory bulb in cochlear nucleus exposed to an augmented acoustic environment.

The regeneration of the auditory neural system remains a challenge in hearing restoration. Acoustic signals may induce a site-specific cell replacement in the auditory system. This hypothesis was tested with grafted implantation of neural precursor cells (NPCs) along the cochlear nucleus in the adult host followed by an augmented acoustic stimulation. NPCs were obtained from the olfactory bulbs at embryonic day 14-16 and were transplanted into the inside border of cochlear nucleus. The labeled cells survived at least 2 weeks, verified by Hoechst 33342 fluorescence, and by immunostaining for a neuronal marker. In some cases NPCs had migrated directionally to the root of the auditory nerve. This observation demonstrates the survival and migration of NPCs from the olfactory bulb (OB) along the adult auditory nerve in an augmented acoustic environment following implantation.

Transplantation of conditionally immortal auditory neuroblasts to the auditory nerve.

Cell transplantation is a realistic potential therapy for replacement of auditory sensory neurons and could benefit patients with cochlear implants or acoustic neuropathies. The procedure involves many experimental variables, including the nature and conditioning of donor cells, surgical technique and degree of degeneration in the host tissue. It is essential to control these variables in order to develop cell transplantation techniques effectively. We have characterized a conditionally immortal, mouse cell line suitable for transplantation to the auditory nerve. Structural and physiological markers defined the cells as early auditory neuroblasts that lacked neuronal, voltage-gated sodium or calcium currents and had an undifferentiated morphology. When transplanted into the auditory nerves of rats in vivo, the cells migrated peripherally and centrally and aggregated to form coherent, ectopic 'ganglia'. After 7 days they expressed beta 3-tubulin and adopted a similar morphology to native spiral ganglion neurons. They also developed bipolar projections aligned with the host nerves. There was no evidence for uncontrolled proliferation in vivo and cells survived for at least 63 days. If cells were transplanted with the appropriate surgical technique then the auditory brainstem responses were preserved. We have shown that immortal cell lines can potentially be used in the mammalian ear, that it is possible to differentiate significant numbers of cells within the auditory nerve tract and that surgery and cell injection can be achieved with no damage to the cochlea and with minimal degradation of the auditory brainstem response.

Development of spontaneous miniature EPSCs in mouse AVCN neurons during a critical period of afferent-dependent neuron survival.

During a critical period prior to hearing onset, cochlea ablation leads to massive neuronal death in the mouse anteroventral cochlear nucleus (AVCN), where cell survival is believed to depend on glutamatergic input. We investigated the development of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in AVCN neurons using whole cell patch-clamp techniques during [postnatal day 7 (P7)] and after (P14, P21) this critical period. We also examined the effects of unilateral cochlea ablation on mEPSC development. The two main AVCN neuron types, bushy and stellate cells, were distinguished electrophysiologically. Bushy cell mEPSCs became more frequent and faster between P7 and P14/P21 but with little change in amplitude. Dendritic filtering of mEPSCs was not detected as indicated by the lack of correlation between 10 and 90% rise times and decay time constants. Seven days after cochlea ablation at P7 or P14, mEPSCs in surviving bushy cells were similar to controls, except that rise and decay times were positively correlated (R = 0.31 and 0.14 for surgery at P7 and P14, respectively). Consistent with this evidence for a shift of synaptic activity from the somata to the dendrites, SV2 staining (a synaptic vesicle marker) forms a ring around somata of control but not experimental bushy cells. In contrast, mEPSCs of stellate cells showed few significant changes over these ages with or without cochlea ablation. Taken together, mEPSCs in mouse AVCN bushy cells show dramatic developmental changes across this critical period, and cochlea ablation may lead to the emergence of excitatory synaptic inputs impinging on bushy cell dendrites.

A disorganized innervation of the inner ear persists in the absence of ErbB2.

ErbB2 protein is essential for the development of Schwann cells and for the normal fiber growth and myelin formation of peripheral nerves. We have investigated the fate of the otocyst-derived inner ear sensory neurons in the absence of ErbB2 using ErbB2 null mutants. Afferent innervation of the ear sensory epithelia shows numerous fibers overshooting the organ of Corti, followed by a reduction of those fibers in near term embryos. This suggests that mature Schwann cells do not play a role in targeting or maintaining the inner ear innervation. Comparable to the overshooting of nerve fibers, sensory neurons migrate beyond their normal locations into unusual positions in the modiolus. They may miss a stop signal provided by the Schwann cells that are absent as revealed with detailed histology. Reduction of overshooting afferents may be enhanced by a reduction of the neurotrophin Ntf3 transcript to about 25% of wild type. Ntf3 transcript reductions are comparable to an adult model that uses a dominant negative form of ErbB4 expressed in the supporting cells and Schwann cells of the organ of Corti. ErbB2 null mice retain afferents to inner hair cells possibly because of the prominent expression of the neurotrophin Bdnf in developing hair cells. Despite the normal presence of Bdnf transcript, afferent fibers are disoriented near the organ of Corti. Efferent fibers do not form an intraganglionic spiral bundle in the absence of spiral ganglia and appear reduced and disorganized. This suggests that either ErbB2 mediated alterations in sensory neurons or the absence of Schwann cells affects efferent fiber growth to the organ of Corti.

Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion.

Time lapse video recordings of cultured adult human and guinea pig spiral ganglion (hSG and gpSG) show that mitogen responsive progenitor/stem cells develop in the form of spheres that proliferate and differentiate into mature neurons and glia cells. Neurospheres, cultured with EGF and bFGF showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). Newly formed BrdU labelled cells were positive for beta-tubulin, and also for GFAP demonstrating that neuronal cells were derived from a dividing population of progenitor cells. Dissociated spheres cultured either with glia cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), induced differentiation of the progenitor cells. Video microscopy showed that neurons develop from subcultured spheres maintained for up to four weeks. Neurons showed fasciculation and migration with a speed of 10-30 microm/h, and some cells had up to 6 mm long neurites coexpressing TrkB and TrkC receptors. Precise dissection suggests that the neurons formed are cochlea-specific. The results suggest that the mammalian auditory nerve has the capability for self-renewal and replacement. Transplantation of progenitor cells together with established means to induce neural differentiation and fiber growth may facilitate strategies for better repair and treatment of auditory neuronal damage.

Electrical stimulation of the cochlear nerve in rats: analysis of c-Fos expression in auditory brainstem nuclei.

We investigated functional activation of central auditory brainstem nuclei in response to direct electrical stimulation of the cochlear nerve using c-Fos immunoreactivity as a marker for functional mapping. The cochlear nerve was stimulated in the cerebellopontine angle of Lewis rats applying biphasic electrical pulses (120-250 muA, 5 Hz) for 30 min. In a control group, bilateral cochlectomy was performed in order to assess the basal expression of c-Fos in the auditory brainstem nuclei. The completeness of cochlear ablations and the response of auditory brainstem nuclei to electrical stimulation were electrophysiologically verified. C-Fos immunohistochemistry was performed using the free floating method. In anaesthetized animals with unilateral electrical stimulation of the cochlear nerve, increased expression of c-Fos was detected in the ipsilateral ventral cochlear nucleus (VCN), in the dorsal cochlear nucleus bilaterally (DCN), in the ipsilateral lateral superior olive (LSO) and in the contralateral inferior colliculus (IC). A bilateral slight increase of c-Fos expression in all subdivisions of the lateral lemniscus (LL) did not reach statistical significance. Contralateral inhibition of the nuclei of the trapezoid body (TB) was observed. Our data show that unilateral electrical stimulation of the cochlear nerve leads to increased expression of c-Fos in most auditory brainstem nuclei, similar to monaural auditory stimulation. They also confirm previous studies suggesting inhibitory connections between the cochlear nuclei. C-Fos immunoreactivity mapping is an efficient tool to detect functional changes following direct electrical stimulation of the cochlear nerve on the cellular level. This could be particularly helpful in studies of differential activation of the central auditory system by experimental cochlear and brainstem implants.

Morphological relationships of peptidergic and noradrenergic nerve terminals to olivocochlear neurones in the rat.

In the rat, the outer hair cells in the cochlea receive direct synaptic input from neurones in the ventral nucleus of the trapezoid body. These so-called medial olivocochlear neurones exert an inhibitory influence on the cochlear neural output. Electrophysiological in vitro studies suggest that the activity of medial olivocochlear neurones may be affected by a variety of neuropeptides as well as noradrenaline, but anatomical confirmation of direct synaptic input is still lacking. We have investigated, at the light microscopical level, the morphological relationships between terminals containing noradrenaline, substance P, cholecystokinin and leu-enkephalin, and medial olivocochlear neurones in the rat. A retrograde tracer was injected into the cochlea to label medial olivocochlear neurones and a double labelling immunocytochemical method was used to visualise the retrograde tracer as well as the neurotransmitters within each brain section. Light microscopical analysis revealed nerve endings containing substance P, cholecystokinin and leu-enkephalin in close apposition to the dendrites of medial olivocochlear neurones, and nerve endings containing dopamine-beta-hydroxylase, a marker for noradrenaline, in close contact with the somata as well as dendrites of medial olivocochlear neurones. Although the technique cannot prove the existence of functional synaptic contacts, the results are broadly consistent with electrophysiological data and suggest a direct input to medial olivocochlear neurones from substance P, cholecystokinin, leu-enkephalin and noradrenaline-containing neural pathways. Differences in the densities and spatial distribution of the various neuropharmacological inputs suggest differences in the relative strengths and possible roles of these diverse inputs to the olivocochlear system.

Expression of Na, K-ATPase alpha and beta isoforms in the neonatal rat cochlea.

The postnatal expression of five Na, K-ATPase alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) subunit isoforms in the rat cochlea was investigated by immunocytochemistry. High levels of expression of the alpha 1 and beta 2 isoforms were observed in stria vascularis (SV) at all developmental stages. alpha 1 and beta 1 isoforms showed a distinct time-dependent developmental expression pattern in tissues of the spiral ligament (SL) and spiral limbus (SLi). Limited, temporary expression of alpha 2 and alpha 3 subunit isoforms were found in SV and SL. Expression of each isoform was also seen in organ of Corti (OC), spiral ganglion (SG), cochlear nerve (CN) and Kölliker's Organ (KO). These observations suggest that individual isoforms may exert specific actions postnatally during final cochlear maturation.