Doctoral (Ph.D.) Thesis

NEW OBSERVATIONS ON THE ANATOMY OF THE OLIVOCOCHLEAR SYSTEM AND AUDITORY PATHWAY

Dr. Miklós Horváth

Tutors: Prof. Dr. Miklós Palkovits and Prof. Dr. Ottó Ribári

Ph.D. School of the Semmelweis University Ph.D. School of Molecular Medicine (7) Program in Pathobiochemistry (7/2) Head of the Program: Prof. Dr. József Mandl

Budapest, 2003 1. Introduction

On the lowest level of the descending , a massive projection originates in the of the to innervate outer and inner hair cells, or their immediate afferent , in the . This olivocochlear system shares its developmental origin with facial branchial motor neurons and may be considered to be of the special visceral efferent type. The olivocochlear system was earlier considered to consists of two main populations of cells in the superior olivary complex. The medial olivocochlear cells (MOCs) reside in the ventral nucleus of the of both sides of the brain and innervate outer hair cells in the cochlea. Lateral olivocochlear cells (LOCs) are associated to the lateral nucleus of the superior olive (LSO), innervate predominantly the ipsilateral cochlea and terminate beneath the inner hair cells. The lateral olivocochlear neurons can be further divided into two subgroups. Small and fusiform cells are located within the ipsilateral LSO are termed intrinsic lateral olivocochlear neurons, while larger olivocochlear neurons are found in the periolivary regions around the lateral nucleus of the superior olive of both sides extending dendrites into the LSO and may be called shell neurons or shell LOCs. The collateral projections of olivocochlear cells were extensively studied in gerbil, mouse and guinea pig, but several controversies still exist about their pattern. Ryan reported that the unmyelinated intrinsic LOCs innervate mainly the central, and the myelinated MOCs the peripheral part of the ventral in gerbil. By contrast others found no cochlear nucleus collaterals of LOCs in gerbil, mouse and cat, but a majority of MOC axons was found to give off collaterals to granule cell containing regions of the . White and Warr showed, by injecting horseradish peroxidase into the cochlea, that axons of olivocochlear cells leave the olivocochlear bundle at several points and enter the dorsal and ventral cochlear nuclei in rat. Position and number of cell bodies that belong to these collaterals have not been evaluated in detail. The nuclei of the ascending and descending auditory pathways are interconnected through a series of commissural and ipsilateral connections. Each stage of the descending auditory pathway could serve as descending part of regional feedback loops. In contrast to this “loop hypothesis”, some studies have indicated that descending auditory fibers form a continuous chain of neurons extending from the to the . To gain more insight into the nature of the descending auditory system several studies have investigated the possible sources of neuronal inputs to olivocochlear cells. The combination of retrograde and anterograde tracers could show some specific synaptic inputs to the olivocochlear cells.

2 Physiological and anatomical data indicate direct cortical innervation of olivocochlear cells. Descending projections from the were also shown to terminate on olivocochlear neurons. Lateral olivocochlear neurons are innervated by cells of the ipsilateral posteroventral cochlear nucleus, while medial olivocochlear neurons receive inputs from the posteroventral and anteroventral cochlear nucleus bilaterally. In addition to these auditory connections, considerable evidence suggests the existence of peptidergic, noradrenergic and serotonergic inputs to olivocochlear neurons. New transneuronal tracing techniques have been developed, based on the use of alpha- herpesviruses (herpes simplex virus type 1 and pseudorabies virus) and rabies virus. These methods make it possible to identify functional networks of connected neurons. Following injection into peripheral organs or directly into the central nervous system, the viruses serve as self-amplifying markers. Virus-infected neurons can be visualized by routine immunocytochemical techniques. While the nervous system matures, the nerve cells go through a series of stages, each of which is characterized by a specific group of active genes and the expression of a specific set of proteins. One marker for distinctive developmental stages is the growth associated protein GAP-43. The protein GAP-43 (also called B-50, F1, pp46, P-57, or neuromodulin) is a membrane-associated phosphoprotein enriched in elongating axons and growth cones and it undergoes fast axonal transport in regenerating neurons. GAP-43 induces filopodia in non- neuronal cells and may directly contribute to growth cone activity by regulating cell membrane structure. It is a major substrate of the protein kinase C family and was found to be correlated with long-term synaptic potentiation in the rat hippocampus. With the onset of process outgrowth, intense GAP-43 immunoreactivity appears along the whole length of axons. At the end of this period of intense axonal staining, there is a brief interval in which high levels of GAP-43 immunostaining are seen in the neuropil. This period of dense neuropil staining coincides with the formation of axonal end-arbors, the beginning of synaptogenesis, and the time at which synaptic organization can be modified by the impinging pattern of activity. Later in development, staining in neuropil declines sharply in most regions except for certain structures in the rostral neuraxis which may be sites of ongoing synaptic remodeling. Apart from its role in development, however, it was shown that synaptic remodeling in the adult animal again raises levels of GAP-43 expression. GAP-43 is also part of the molecular machinery underlying long term potentiation, a cellular process related to learning and memory. In the adult animal, re-emergence of GAP-43 has been seen, for instance, in axotomized sensory and motor neurons.

3 2. Aims

2. 1. First, we attempted to monitor auditory brainstem development in the rat by using GAP- 43 as a developmental marker. The aim of our work was to determine the expression of GAP- 43 in the superior olive and cochlear nucleus in the early postnatal period and in adult animals.

2. 2. Second, we attempted to learn in detail, what kinds of changes occur in GAP-43 expression of the auditory brainstem after lesions to the . Using GAP-43 as a marker for synaptogenesis and plasticity, we wanted to determine the plastic changes in the superior olive and cochlear nucleus following the loss of their inputs from the auditory periphery.

2. 3. Third, experiments were done to determine the distribution and number of olivocochlear cells that give off collaterals into the ventral cochlear nucleus en route to the cochlea in rat. The retrograde axonal tracers Diamidino Yellow and Fast Blue were used to label cell bodies of individual olivocochlear neurons and visualize cells that project to the cochlear nucleus.

2. 4. Fourth, the aim of our work was the transneuronal labeling of the descending auditory system by injecting the pseudorabies virus directly into the cochlea. This method allowed us to identify the olivocochlear cells and the sources of their inputs from other auditory and non- auditory neurons.

3. Methods

3. 1. To examine the postnatal development of GAP-43 immunoreactivity in the cochlear nucleus and superior olive, 30 Wistar rats were reared with their mothers with food and water available ad lib. Young animals were anaesthetized with lethal doses of barbiturate and their brains were removed and placed in ice-cold fixative. Older rats were killed by an overdose of Nembutal (Abbott) and perfused transcardially with 1 l ice-cold fixative for 1 hour. Thirty micron frontal or sagittal sections, cut on a cryostat, were collected in phosphate buffer. Immunocytochemical detection of GAP-43 was achieved with an antibody from mouse and a biotinylated anti-mouse immunoglobulin G secondary antibody. Peroxidase labeling was

4 visualized by using diaminobenzidine and ammonium nickel sulfate. Control sections that were incubated by omitting the primary antibody never showed staining of specific cellular elements. Great care was taken to keep the parameters influencing the outcome of the staining constant throughout the whole experimental series.

3. 2. Twenty-nine Wistar rats of both sexes were used to analyze the changes in GAP-43 expression of the auditory brainstem, following the removal of the left cochlea. The animals were deeply anaesthetized, the bulla tympani was approached and opened. The bony wall of the cochlea was perforated with a spherical drill head and the interior of the cochlea, including the spiral ganglion, was cleared. After different survival times (2, 4, 7, 14, 28, 56 days), animals were reanaesthetized with lethal doses of Nembutal and perfused transcardially with 1 l ice-cold fixative. Brains were cut on a cryostat into 30 µm thick frontal sections. Subsequently, sections were exposed to an anti-GAP-43 antibody, which is specified as recognizing GAP-43 independent of its state of phosphorylation. After incubation times of 72 hours at 4°C, binding-sites of the anti-GAP-43 antibody were detected using the avidin- biotin-technique with diaminobenzidine and ammonium nickel sulfate to intensify the colour of the reaction product. Parallel sections were stained for glutamate and calretinin immunoreactivity to observe integrity or degree of disintegration of the at its entrance into the brainstem. Some sections were incubated with an antibody against synaptophysin. In order to succeed with an at least limited quantitative approach, every effort was made to keep conditions constant for the complete experimental procedure. Duration and temperature of perfusion, interval between perfusion and exposure to primary antibody, incubation times, temperature and concentration of primary and secondary antibodies and of chemicals for the DAB-staining reaction were unchanged during the whole experimental series.

3. 3. Sixteen adult Wistar rats of both sexes were used to determine the distribution and number of olivocochlear cells that give off collaterals into the ventral cochlear nucleus en route to the cochlea. Two kinds of operations were done. In the first group (n=6), the left cochlea was approached by a lateral opening of the tympanic bulla. A small hole was drilled into the cochlear wall, centered over the middle of its long axis and part of the perilymph was soaked off using small tissue wicks. Fast Blue (n = 4) or DY (n = 2) was tamped into the cochlea and the opening closed with surgical bone wax. The bulla was subsequently filled with Gelfoam and the wound surgically closed. In the second group (n=10), FB was injected

5 into the left ventral cochlear nucleus (VCN), immediately followed by application of DY into the cochlea of the same side. To approach the VCN, a small craniotomy was made in the left occipital bone and the overlying cerebellar flocculus and paraflocculus were aspirated to expose the cochlear nucleus. Using pressure, 0.15 ? l saturated solution of FB were slowly injected into the ventral cochlear nucleus. The application of DY into the cochlea was done as described for the first group of animals. The animals were allowed to recover for a period of 5 or 6 days. They were then deeply anaesthetized and fixed by transcardial perfusion with 4 % paraformaldehyde in phosphate buffer. Thirty micrometer thick frozen sections of the brainstem were cut in the frontal plane and mounted on gelatin-subbed slides. Sections were viewed with a Zeiss microscope using epi-illumination. The exciting filter used had a peak at 405 nm and the barrier filter cut off was at 455 nm. To localize the injection site and the labeled cells within the various brainstem nuclei, maps were made based on camera lucida drawings. Individual olivocochlear neurons were examined using a 100x oil-immersion objective in order to distinguish single from double-labeled cells. The number of labeled cells were determined using profile counts.

3. 4. Seventeen adult male guinea pigs were utilized to identify the olivocochlear cells and the sources of their inputs from other auditory and non-auditory neurons. Bartha´s strain of Aujeszky's disease virus (taxonomic name: suid herpesvirus 1; commonly used name: pseudorabies virus; PrV-Ba) was propagated in monolayers of porcine kidney (PK15) cells, and assayed by a standard plaque assay. Aliquots containing 109 pfu/ml (plaque-forming units/ml) were used in the experiments. The animals were anaesthetized, the left tympanic bulla was opened and the round window was exposed. A suspension of 50 µl of the virus was slowly injected into the cochlea, through the round window. After completing the injection, the round window was closed with surgical bone wax. The guinea pigs were sacrificed 15 h (n=1), 25 h (n=2), 37 h (n=3), 40 h (n=2), 48 h (n=3), 72 h (n=6), 90 h (n=2), 96 h (n=1), 108h (n=1) after the viral injection. Animals were anesthetized again and perfused transcardially with fixative solution. The brains were removed from all animals, immersion-fixed for 6 hours in the same fixative solution. The virus-infected cells were visualized by immunoperoxidase staining of cryosections (50 µm thick) Sections were collected in two series from which one was treated for virus immunolabeling. The immunhistological procedure was carried out on free-floating sections. The sections were incubated with the primary antiserum for 24-48 hours and the antibody was visualized with avidin/biotin peroxidase staining by the ABC technique. Sections were

6 mounted, counterstained with cresylviolet and coverslipped. The structures of the brain were identified using the stereotaxic atlas of the guinea pig brain.

4. Results

4. 1. The neonatal superior olivary complex and cochlear nucleus showed high levels of GAP- 43 immunoreactivity. This immunoreactivity gradually decreased until, in the adult, comparatively low levels of GAP-43 immunoreactivity were left. The light microscopical analysis of the staining pattern at high power revealed that neonatally, GAP-43 immunoreactivity is diffusely distributed in the neuropil. Over the first two postnatal weeks, GAP-43 gradually changed from this dense diffuse distribution to a more granular distribution of reduced intensity. Eventually, GAP-43 became localized in distinctly delineated varicose fibers in the adult animals. In the anteroventral cochlear nucleus, immunoreactive boutons were often located at perikaryal surfaces.

4. 2. Building on the knowledge that fibers of the cochlear nerve contain glutamate as well as calretinin, we stained the cochlear nucleus of operated animals at varying postoperative times for these substances after cochlear ablation. Degeneration of nerve fibers on the operated side was readily detectable as early as on the 4. postoperative day in both stains, and the loss of primary auditory fibers was still apparent after two months, indicating that the auditory brainstem was indeed deprived from ipsilateral cochlear input. As a consequence of removal of the , GAP-43 immunoreactivity in VCN, which is low in normal adult rats, rose dramatically on the operated side. Moreover, GAP-43 expression also increased in the superior olivary nucleus. Analysis of GAP-43 immunoreactivity in the cochlear nucleus revealed a specific sequence of events triggered by cochlea destruction. By the 2. postoperative day, GAP-43 immunoreactivity has not yet changed as compared to the unaffected contralateral side. The control level of staining consisted in a few but distinctly defined bouton-like structures, about 1 to 2.5 µm wide. By the 4. postoperative day, dense GAP-43 staining appeared in the neuropil. This staining was mostly accumulated in axonal swellings while immunoreactive cell bodies were not observed. It was particularly rich in the vicinity of neuronal perikarya, and a large number of unstained cell bodies were seen embedded in the immunopositive neuropil. The intensity of GAP-43 immunoreactivity reached a climax around postoperative day 7. Two weeks after the lesion, GAP-43 immunoreactivity was clearly weaker than a week earlier. In addition to this neuropil staining,

7 the contralateral dorsal cohlear nucleus contained some GAP-43-positive cells. These immunopositive cell bodies had very different sizes and morphologies. By sharp contrast to the cochlear nucleus, the major postoperative increase of GAP-43 immunoreactivity occurred not in the neuropil, but in cell bodies of the superior olivary complex. GAP-43 immunoreactivity rose in cell bodies of the lateral superior olivary nucleus (LSO) to a maximum on postoperative day 7, to decrease thereafter.

4. 3. Following FB or DY application into the cochlea, retrogradely labeled cells were encountered bilaterally in the SOC as described earlierSmall (diameter approximately 10 ? m) and fusiform neurons inside the LSO (intrinsic LOCs) were labeled virtually exclusively on the ipsilateral side, whereas the majority of larger (approximately 15 ? m) multipolar cells of the VNTB (MOCs) resided on the contralateral side. Additionally, larger (about 15 ? m) neurons in the periolivary region around LSO were labeled with an ipsilateral dominance. The total number of labeled olivocochlear cells was 901.0 ? 17.7 S.E.M. after tracing with FB, and 1129.0 ? 13.0 S.E.M. after DY application. In the second series of experiments we combined the retrograde labeling of olivocochlear cells with the visualization of cells that project to the VCN. Diamidino Yellow was applied to the left cochlea to trace olivocochlear cells and FB was injected into the VCN of the same side. Following the FB injection into VCN, a large number of periolivary cells were labeled, showing an ipsilateral dominance. Principal neurons of the ipsilateral medial nucleus of the trapezoid body were also labeled. The ipsilateral LSO had a few (8.8 ? 2.3 S.E.M.), the contralateral had no labeled neurons. Additionally, multipolar neurons of the contralateral VCN, some cells in the deeper layers of the contralateral DCN, and neurons of all three subnuclei of the inferior colliculus were also labeled. The combination of DY and FB allowed us to identify olivocochlear cells giving off collaterals into VCN. The combined application of these tracers resulted in partly intermingled populations of blue and/or yellow labeled neurons. Single labeled neurons showed only yellow fluorescence of the nucleus or blue staining of the cytoplasm. Double labeled neurons were clearly distinguishable by the intense yellow fluorescence of their nucleus and their blue cytoplasm. We found double-labeled cells in the periolivary region around LSO (shell LOCs) and in the VNTB (MOCs) of both sides of the brain. Across five experiments, only six double-labeled neurons were found in the ipsilateral LSO. Fifty-six percent of the MOCs in the VNTB and 39% of shell neurons were seen by virtue of their double labeling to send collaterals into the VCN. These percentages resulted from retrograde

8 transport originating in a restricted area of VCN and are expected to grow, possibly up to 100%, with a larger or even complete coverage of VCN with tracer, which is difficult to achieve for practical reasons.

4. 4. There were no virus-infected neurons visible in the brain 15 hours (n=1) after the intracochlear injection of the pseudorabies virus. After longer survival periods of time (25- 108 hours, n=16) labeled cells were seen in the superior olive, in other auditory brainstem nuclei bilaterally, in the auditory cortex and in several monoaminergic regions. On the basis of labeled regions, three stages of infection were distinguished. Stage 1 (25 hours) was characterized by infection of the olivocochlear cells, while stage 2 (37-72 hours) was identified by labeling in the inferior colliculus and in other auditory and non-auditory structures of the brainstem. Following longer survival times (stage 3, 90-108 hours) infected cells were additionally seen in the medial geniculate body and auditory cortex bilaterally. The first labeled neurons were observed in the ipsilateral lateral superior olive (LSO) and in periolivary regions bilaterally at 25 hours survival time. Fibers and small (10 to 12 ? m) fusiform neurons were labeled in the ipsilateral LSO, while some larger infected neurons appeared at the dorsomedial and dorsolateral border of this nucleus. Larger (15-20 ? m) multipolar cells of the superior olivary complex were also labeled by the virus on both sides. They were localized in the ventral nucleus of the trapezoid body (VNTB) and in rostral periolivary regions (RPO). About two-thirds of the labeled cells were found contralateral to the virus-injected cochlea and one third of them were ipsilaterally. These medial olivocochlear neurons formed a continuous cell column from the VNTB through the RPO, up to the ventral nucleus of the . The retrograde infection of the olivocochlear efferent cells was still not equally effective in all animals sacrificed between 37 and 72 hours after viral application. In ten of fourteen brains, infected olivocochlear cells were found in the ipsilateral LSO, in the VNTB and RPO bilaterally. Labeled neurons were also noted in other nuclei of the auditory brainstem indicating the transneuronal propagation of the virus. Both the ventral and dorsal nuclei of the lateral lemniscus contained labeled cells bilaterally. The central nucleus of the inferior colliculus showed a fairly high number of infected cells. The labeled cells were found bilaterally with a small contralateral dominance. No labeled cells were seen in the inferior colliculus outside the central nucleus. Very few multipolar neurons were labeled in the ventral cochlear nucleus bilaterally with a slight contralateral dominance. Some scattered infected

9 cells could be detected bilaterally in the medial . In addition to the auditory brainstem, neurons of some brainstem monoaminergic cell goups were abundantly labeled at 37-72 hours survival times. A high number of cells were labeled in the pontine dorsal raphe and neurons in the locus coeruleus and the subcoeruleus area were also bilaterally labeled. Following longer survival times (90-108 hours, n?4), virus infected cells were found all along in nuclei of the auditory brainstem and in brainstem monoaminergic regions. Infected cells were also found in the medial geniculate body and in the primary auditory cortex bilaterally. In the medial geniculate body, more labeled cells were found in the ventral than in the dorsal subdivision. Although the number of labeled cells was not quantified, the contralateral medial geniculate body seemed to contain more infected neurons than the ipsilateral. The labeled neurons in the auditory cortex were restricted to layer V and were pyramidal in shape.

5. Conclusions

5. 1. The data collected in this work indicate that critical periods of auditory system ontogeny are likely to fall into the time window of massive synaptic maturation in the auditory brainstem nuclei as visible through GAP-43 immunocytochemistry. We suggest that the most pronounced changes in the pattern of GAP-43 staining occurring between P8 and P16 take place during major critical developmental periods. It appears as if the transition from a predominantly homogeneous distribution of GAP-43 to a predominantly granular localization mainly falls into the time period between P8 and P16, suggesting that this is the period when the majority of axons turn from growth-cone tipped fibers to processes ending in presynaptic boutons. The emerging granules in the pattern of GAP-43 staining must have formed in part before stimulus-dependent activation of spiral ganglion cells reaches the cochlear nucleus, while a second phase of synaptic maturation in the cochlear nucleus takes place with cochlear nerve activity driven by sensory stimuli.

5. 2. The dynamics of GAP-43 re-expression in the ventral cochler nucleus and superior olive after cochlear lesion underline the prominence and significance of synaptic reorganizations following alterations of the auditory input. Ablation of the cochlea has severe effects on the auditory brainstem nuclei, resulting in modifications of connectivity and, consequently, neuronal communication. Unilateral cochlear ablation does three things to the auditory system. It deprives the system from sensory input from one side, it induces degenerative processes that affect all cochlear nerve fibers directly and their targets in the cochlear nucleus

10 indirectly, and it axotomizes neurons of the olivocochlear bundle. The changes in neuronal structure induced by cochlear ablation are the sum of these processes. The potential for plastic reactions of the adult auditory system to an altered input is mostly attributed to the rostral, or higher, auditory structures, particularly the auditory cortex, the medial geniculate body, and the superior colliculus. Our results indicate that the auditory brainstem nuclei play an important role in plastic reactions of the auditory system.

5. 3. The major results of the tracing experiments with the fluorescent tracers are that (1) the intrinsic lateral olivocochlear neurons in the LSO of the rat do not have axon collaterals reaching into VCN; that (2) a significant number, and possibly a majority, of shell neurons around the LSO of both sides give off collaterals into VCN and, by virtue of their specific connectivity, constitute a distinct population of olivocochlear cells; and that (3) a majority of MOCs, but possibly even all of them, residing in the VNTB of both sides, send axon collaterals into VCN and are therefore in a position to massively influence the ascending auditory pathway not only at the level of the hair cells, but also on the level of the cochlear nucleus. The massive collateral projection of MOCs found in the present study indicates that MOCs must play a crucial role in feedback control of auditory processing. These cells can directly influence the motility of OHCs as well as the secondary sensory neurons in the cochlear nucleus and are themselves under the influence of an excitatory input from the contralateral cochlea by way of the cochlear nucleus, thus serving to link both cochlea and both cochlear nuclei. This regional feedback loop, together with descending pathways from the inferior colliculus and auditory cortex constitutes the anatomical basis for the efferent control of auditory processing in the lower brainstem.

5.4. The present work indicate that the descending auditory system is not only a loosely interconnected chain of feedback loops, but also a descending chain of neurons which is able to conduct information from the auditory cortex to the cochlea. Applying traditional tracer substances, it was not possible to identify the existence of the descending chain of neurons from the auditory cortex to the inner ear. After the intracochlear application of the pseudorabies virus, olivocochlear neurons became infected and after longer survival times viruses reached higher auditory related structures. The descending chain consist of pyramidal cells in layer V of the auditory cortex, neurons of the medial geniculate body (ventral subdivision), inferior colliculus (central nucleus), nuclei of the lemniscus lateralis (dorsal and ventral), olivocochlear neurons and hair cells of the cochlea. Thus, the activity of many

11 neurons in the central and peripheral auditory system can be affected by descending projections. Serotonin was early shown to be present in the cochlear nuclei, superior olivary complex and nuclei of the lemniscus lateralis. Serotonergic projections to olivocochlear neurons were firstly described by Thompson and Thompson. Woods and Azeredo also showed numerous serotonergic boutons in contact with labeled olivocochlear cells. The sources of these serotonergic inputs were previously not determined. We found many virus-infected cells at stage 2 and later in the pontine part of the dorsal raphe nucleus, suggesting that serotonergic inputs to olivocochlear cells arise in this region. Noradrenergic inputs to the auditory brainstem were also shown in several species. Experiments by using combined retrograde and anterograde tracing and immunofluorescence method indicated that noradrenergic fibers to the olivocochlear neurones arise in the locus coeruleus. In the present study, the localization of virus-infected cells at stage 2 confirms this observation and suggests that noradrenergic neurons innervating olivocochlear cells are located in the locus coeruleus and even more numerous in the subcoeruleus area. It is more likely, that noradrenergic and serotonergic neurons directly modulate the activity of the olivocochlear neurons, and thus alter cochlear potential.

6. Acknowledgments

I wish to express my gratefulness to my tutors, Prof. Miklós Palkovits and Prof. Ottó Ribári for their unselfish and steady help. I gratefully acknowledge the valuable contributions of all employees of the Laboratory of Neuromorphology, Semmelweis University. I thank the generous support of Prof. Robert B. Illing and all colleagues in the Neurobiological Laboratory of the Department of Otolaryngology, University Freiburg. I have spent two useful years at this Department in Freiburg. I thank the valuable support of Prof. Gábor Répássy, Head of the Department of Otolaryngology, Head and Neck Surgery, Semmelweis University Budapest. I am grateful to my family for their help and understanding.

12 7. Publiations and abstracts of the author in connection with the present work

Publications:

Illing RB, Horváth M. 1995. Re-emergence of GAP-43 in cochlear nucleus and superior olive following cochlear ablation in the rat. Neuroscience Letters 194: 9-12.

Horváth M, Förster CR, Illing RB. 1997. Postnatal development of GAP-43 immunoreactivity in the auditory brainstem of the rat. Journal of Comparative Neurology 382: 104-115.

Illing RB, Horváth M, Laszig R. 1997. Plasticity of the auditory brainstem: effects of cochlear lesions on GAP-43 expression in rat. Journal of Comparative Neurology 382: 116-138.

Horváth M, Kraus KS, Illing RB. 2000. Olivocochlear neurons sending axon collaterals into the ventral cochlear nucleus of the rat. Journal of Comparative Neurology 422: 95-105.

Horváth M, Ribári O, Répássy G, Tóth IE, Boldogkoi ZS, Palkovits M. 2003. Intracochlear injection of pseudorabies virus labels descending auditory and monoaminerg projections to olivocochlear cells in guinea pig. European Journal of Neuroscience, accepted for publication.

Abstracts:

Förster CR, Horváth M, Illing RB. 1995. Postnatal development and reemergence of GAP-43 in auditory brainstem nuclei following cochlear lesioning. European Journal of Neuroscience, Suppl. 8: p. 34.

Illing RB, Horváth M, Michler S, Laszig R. 1996. Gibt es inhibitorische Synapsen an Neuronen der Cochlea? HNO Informationen, 21: 99.

Illing RB, Förster CR, Horváth M. 1996. Evaluating the plasticity potential of auditory brainstem nuclei in the rat. American Journal of Otology, Suppl. 6: pp. 52-53.

Michler S, Illing RB, Häufel T, Horváth M, Laszig R. 1999. Audiometrie der Ratte: Hörvermögen und Ertaubungsmodelle. HNO, 47: 413.

13 Michler S, Häufel T, Horváth M, Illing RB. 1999. Three different models of monaural deafness of the rat: brainstem audiometry and histology. Proceedings of the 1st Göttingen Conference of the German Neuroscience Society pp. 294.

Horváth M, Kraus KS, Illing RB, Laszig R. 2000. Medial olivocochlear neurons innervate the ventral cochlear nucleus in rat. Laryngo-Rhino-Otologie, Suppl. 79: pp. 119.

Ribári O, Palkovits M, Horváth M. 2001. Kontralaterale Hörverbesserung nach Cochlear Implant: Klinische Ergebnisse und anatomische Grundlagen. HNO Informationen, 25: pp. 167

Presentations in connection with the present work:

Horváth M, Tóth IE, Boldogkoi Zs, Medveczky I, Ribári O, Palkovits M. 1997. Investigation of descending pathways of the auditory system via transsynaptic labelling in guinea pig. VI. Semmelweis Scientific Forum, Budapest.

Ribári O, Küstel M, Horváth M. 1998. Tinnitus controll in cochlear implant patients. 25. International Congress of the Society of Neurootology and Equilibrometry, Bad Kissingen

Horváth M, Tóth IE, Boldogkoi Zs, Medveczky I, Ribári O, Palkovits M. 1998. Labelling of descending pathways of the auditory system via pseudorabies virus. 4. International Congress of the Worldwide Hungarian Medical Academy, Budapest.

Horváth M, Tóth IE, Boldogkoi Zs, Ribári O, Palkovits M. 2000. Innervation of the olivocochlear cells studied by transsynaptic tracing in guinea pig and rat. 4. Congress of the Hungarian Neuroscience Association, Budapest

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