Durch die Überalterung unserer Gesellschaft nimmt die Prävalenz für altersbedingte Hörschäden zu. Eine zunehmende Zahl hochgradig schwerhöriger oder ertaubter Patienten wird daher mit Cochlea-Implantaten (CIs) versorgt. Ungewiss ist bislang, ob es altersabhängige Unterschiede in der Modulierbarkeit des zentralen auditorischen Systems gibt. Dazu erforschten wir die Regulation des Plastizitätsmarkers Gap43 nach CI-Stimulation adult ertaubter Ratten unterschiedlichen Alters.

Plasticity enables the central auditory system to adapt to changes in the sensory-evoked activity such as sudden deafness. We investigated how plastic processes induced in the adult auditory brainstem by unilateral cochleotomy correlate with changes of the extracellular matrix (ECM). We focused on the expression of the plasticity associated protein Gap43 and the lectican neurocan in the rat's cochlear nucleus (AVCN) and inferior colliculus (CIC).

Die Plastizität des Säugergehirns ermöglicht es dem auditorischen System auf Veränderungen des sensorischen Inputs zu reagieren. Wir haben die Zusammenhänge zwischen plastischen Prozessen nach einseitiger Cochleotomie im auditorischen Hirnstamm und Veränderungen der extrazellulären Matrix (EZM) untersucht. Im Fokus standen hierbei der Plastizitätsmarker Gap43 und das Lectican Neurocan im Cochlearis-Kern (AVCN) und im Colliculus inferior (CIC).

As a result of aging, the prevalence of age-related hearing loss rises in our society. An increasing number of profoundly deaf or completely deaf patients are supplied with cochlear implants (CIs). Up to now, it is unknown whether there are age-dependent differences in the plasticity of the central auditory system. For this purpose, we investigated the expression of the plasticity marker Gap43 after CI stimulation of adult rats deafened at different ages.

Expression of the immediate-early gene fos (also known as c-fos) and phosphorylation of the product of the early response gene atf2 (pATF2) in the adult auditory brainstem can be modulated by electrical intracochlear stimulation. The Fos and pATF2 proteins are competitive monomers of the heterodimeric activator protein-1 (AP-1) transcription factor that triggers the expression of genes related to neural plasticity. Our previous findings showed that the stimulation-induced spatio-temporal pattern of Fos expression in the adult auditory system depends on hearing experience. In this study, we aimed to identify a possible correlation of pATF2 and Fos expression. Adult normal hearing and neonatally deafened rats were unilaterally stimulated with a cochlear implant (CI) for 45 min, 73 min, or 2h. The numbers of Fos- and pATF2-positive neurons in the anteroventral cochlear nucleus (AVCN), the lateral superior olive (LSO), and the central inferior colliculus (CIC) were evaluated. Following stimulation, an increased Fos expression was demonstrated in all these regions in hearing and deaf rats. However, in neonatally deafened rats, significantly more Fos-positive neurons emerged that did not obey a tonotopic order. Independent of hearing experience, Fos expression correlated with a locally matching decrease of pATF2 expression in AVCN and LSO, but not in CIC. We suggest that these changes in gene expression result in a shift of AP-1 dimer composition from ATF2:Jun to Fos:Jun. This change in AP-1 constellation is expected to invoke different transcriptional cascades leading to distinct modes of tissue reorganization and plasticity responses in the mature central auditory system under stimulation.

During pre- and postnatal development, a high level of growth-associated protein 43 (Gap43) is expressed in the brain. This neuron-specific protein is expressed in somata, axons, and growth cones and plays a key role in neurite outgrowth and synaptogenesis. With maturation of the brain, Gap43 is down-regulated by most neurons, except in brain areas such as the hippocampal CA3 region or the binaural auditory regions lateral superior olive (LSO) and central inferior colliculus (CIC). This study investigated how changes in sensory activity levels and patterns can modulate the adult plasticity response. To study the effect of sensory activity on adult Gap43 expression, mRNA and protein levels were determined in LSO and CIC of hearing-experienced rats, unilaterally and bilaterally deafened rats, or rats unilaterally stimulated by a cochlear implant (CI). Unilateral hearing loss of an adult auditory system causes asymmetrical expression of Gap43 mRNA between ipsi- and contralateral LSOs or CICs of the brain stem. While the mRNA level rose on the contralateral side of the LSO, CIC neurons increased their gap43 transcription ipsilaterally compared to the control level (p < 0.001). Compensation of the lost sensory input by way of CI stimulation resulted in a bilaterally symmetric but increased gap43 transcription. Our data indicate that Gap43 is not only a marker for neuronal growth and synaptogenesis, but also reflects modified patterns of synaptic activities on auditory neurons. Thus, unilateral deafness directly results in an asymmetrical adaptation of the gap43 transcription between both sides of the auditory brain stem. This can be prevented by simple-patterned stimulation of the auditory nerve via a CI.

Während der frühkindlichen Entwicklung bildet das Gehirn hohe Konzentrationen des wachstumsassoziierten Proteins 43 (Gap43). Als Indikator für Synapsenbildung und Neuritenwachstum wird es in neuronalen Zellkörpern, Axonen und unreifen Synapsen exprimiert, bevor es mit der Reifung des Gehirns vielerorts abnimmt. In der lateralen oberen Olive (LSO) und dem Colliculus inferior (CIC) der zentralen Hörbahn bleibt es jedoch hoch konzentriert. Diese Studie untersucht, inwiefern Änderungen sensorischer Aktivität das durch Gap43 abgebildete adulte Plastizitätspotenzial modulieren können.

We aimed to determine whether rat olivocochlear neurons survive axotomy inflicted through cochlear ablation, or if they degenerate. To estimate their intrinsic potential for axonal regeneration, we investigated the expression of the transcription factor c-Jun and the growth-associated protein-43 (GAP43). Axonal tracing studies based on application of Fast Blue into the cochlea and calcitonin gene-related peptide immunostaining revealed that many, but not all, lateral olivocochlear neurons in the ipsilateral lateral superior olive degenerated upon cochleotomy. A decrease of their number was noticed 2 weeks after the lesion, and 2 months postoperative the population was reduced to approximately one quarter (27–29%) of its original size. No further reduction took place at longer survival times up to 1 year. Most or all shell neurons and medial olivocochlear neurons survived axotomy. Following cochleotomy, 56–60% of the lateral olivocochlear neurons in the ipsilateral lateral superior olive were found to co-express c-Jun and GAP43. Only a small number of shell and medial olivocochlear neurons up-regulated c-Jun expression, and only a small number of shell neurons expressed GAP43. Up-regulation of c-Jun and GAP43 in lateral olivocochlear neurons upon axotomy suggests that they have an intrinsic potential to regenerate after axotomy, but cell counts based on the markers Fast Blue and calcitonin gene-related peptide indicate that this potential cannot be exploited and degeneration is induced instead. The survival of one quarter of the axotomized lateral olivocochlear neurons and of all, or almost all, shell and medial olivocochlear neurons appeared to depend on connections of these cells to other regions than the cochlea by means of axon collaterals, which remained intact after cochleotomy.

When we disturbed the auditory input of the adult rat by cochleotomy or noise trauma on one side, several substantial anatomical, cellular, and molecular changes took place in the auditory brainstem. We found that: (1) cochleotomy or severe noise trauma both lead to a considerable increase of immunoreactivity of the growth-associated protein GAP-43 in the ventral cochlear nucleus (VCN) of the affected side; (2) the expression of GAP-43 in VCN is restricted to presynaptic endings and short fiber segments; (3) axon collaterals of the cholinergic medial olivocochlear (MOC) neurons are the path along which GAP-43 reaches VCN; (4) partial cochlear lesions induce the emergence of GAP-43 positive presynaptic endings only in regions tonotopically corresponding to the extent of the lesion; (5) judging from the presence of immature fibers and growth cones in VCN on the deafened side, at least part of the GAP-43 positive presynaptic endings appear to be newly formed neuronal contacts following axonal sprouting while others may be modified pre-existing contacts; and (6) GAP-43 positive synapses are formed only on specific postsynaptic profiles, i.e., glutamatergic, glycinergic and calretinin containing cell bodies, but not GABAergic cell bodies. We conclude that unilateral deafening, be it partial or total, induces complex patterns of reconnecting neurons in the adult auditory brainstem, and we evaluate the possibility that the deafness-induced chain of events is optimized to remedy the loss of a bilaterally balanced activity in the auditory brainstem.

A unilateral cochlear lesion induces expression of the growth and plasticity‐associated protein 43 (GAP‐43) in fibers and their varicosities on specific types of postsynaptic profiles in the ventral cochlear nucleus (VCN), suggesting the induction of synaptic remodeling. One candidate population from which GAP‐43 might emerge was neurons of the lateral olivocochlear (LOC) system residing in the lateral superior olive (LSO). Upon cochleotomy, these neurons express GAP‐43 mRNA and GAP‐43 protein. However, retrograde axonal tracing with Fast Blue or biotinylated dextran amine from VCN revealed that the number of 6.8 ± 1.3 neurons in the whole ipsilateral LSO labeled in normal adult rats was distinctly small and did not rise after cochleotomy. Concluding that LOC neurons cannot be the source of GAP‐43 in the VCN, we reinvestigated the pattern of GAP‐43 in situ hybridization and found that, after cochleotomy, shell neurons in the regions surrounding the LSO and medial olivocochlear (MOC) neurons in the ventral nucleus of the trapezoid body up‐regulated GAP‐43 mRNA. We then lesioned these regions by means of stereotaxic injections of kainic acid. Destruction of shell neurons preceding an ipsilateral cochleotomy did not change the emergence of GAP‐43 immunoreactivity in the VCN. However, if the contralateral MOC system was lesioned, the rise of GAP‐43 immunoreactivity in VCN on the side of the cochleotomy was significantly reduced. We conclude that, after cochlear dysfunction, MOC neurons are the major (if not exclusive) source of synaptic reorganization in the VCN that could possibly entail compensatory activation of the affected ascending auditory pathway. J. Comp. Neurol. 475:374–390, 2004. © 2004 Wiley‐Liss, Inc.

BACKGROUND:An important factor in the clinical outcome of cochlear implantation is the age of the patient. Compared to older patients, children with congenital deafness have a better outcome when the implantation is made before the age of 2 years. The cause may lie in the molecular biology of the brain, which changes during postnatal maturation.METHODS:Protein probes were obtained from tissue of the rat inferior colliculus at different ages. The probes were analyzed using 2-dimensional SDS electrophoresis.RESULTS:The expression of GAP-43, a protein expressed by neurons during axonal outgrowth and synaptogenesis, and the total number of the protein species showed a significant reduction during ontogenesis. This shows that while neurons gradually assume their specific function, they downregulate GAP-43 and the molecular complexity decreases.CONCLUSIONS:Due to a lack of neuronal pluripotency at later developmental stages, the flexibility to adapt to the afferent activation provided by a cochlear implant is increasingly limited.

Zusammenfassung Hintergrund Die Wichtigkeit des Patientenalters bei Kochleaimplantatversorgung zeigt sich in besonders guten Ergebnissen bei taub geborenen Kindern unter 2 Jahren im Vergleich zu älteren Kindern. Dies könnte in der sich altersabhängig ändernden Molekularbiologie des Gehirns begründet sein. Material und Methoden Proteinproben aus dem Colliculus inferior von Ratten verschiedener Altersstufen wurden mittels zweidimensionaler SDS-Gelelektrophorese analysiert. Ergebnisse Die Expression von GAP-43, einem von Neuronen während des axonalen Wachstums und der Synaptogenese gebildeten Eiweiß, sowie die Gesamtzahl der Proteine nehmen signifikant im Laufe der postnatalen Ontogenese ab—so wie auch die molekulare Komplexität von Neuronen, die spezifische Funktionen annehmen. Schlussfolgerung Infolge mangelnder Pluripotenz der Neuronen reagiert das ältere Gehirn mit verminderter Flexibilität auf die durch das Implantat vermittelten Nervenimpulse.

Studies across mammalian species have indicated that the ontogeny of the central auditory system relies on the utilization of both genetic and environmental instructions. Both types of instructions are relevant for the development of cortical as well as subcortical systems and are apparently used for the establishment of a wide range of functions from cochleotopic representation to language-specific filters in humans. To show this, the pre- and postnatal development of the mammalian central auditory system are briefly considered on seven levels of neuronal organization: neurogenesis, axonal growth, axonal maturation, synaptic maturation, cochleotopy, the descending auditory system, and adult plasticity.

Light microscopy and immunohistochemical analyses of a freshly prepared human cochlea, removed at meningioma skull base surgery, were performed with particular emphasis on synaptophysin (SY) reactivity. Synaptophysin, a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is a useful marker for sites of synaptic transmission of the efferent olivocochlear system in the cochlea. Following fixation and decalcification, cryosections of 30 μm were prepared. To introduce immunostaining, free-floating sections were exposed to monoclonal SY antibody. Positive SY immunostaining was solely restricted to the neural and sensory structures and did not include supporting cells of the organ of Corti. Dense reaction products were noted around the hair cells, especially at the basal portion of the inner and outer hair cells and their neural poles, as well as around the inner spiral bundle, tunnel spiral bundle, outer spiral bundle and upper tunnel crossing fibers. The majority of spiral ganglion cells stained positively. An intermingling network of thin unmyelinated nerve fibers stained densely, especially at the basal portions of the cochlea. The spiral limbus, inner and outer sulcus cells, basilar membrane, myelinated nerve fibers, spiral ligament and the stria vascularis were unstained. Human cochlea obtained during surgery offers excellent conditions for immunohistochemical analysis. In the basal cochlea in the organ of Corti, outer hair cell area, there may be alterations due to noise trauma from the drilling procedure.

We induced acoustic trauma by applying click stimuli of 130 dB (SPL) for 30 min to one ear of adult rats. This treatment resulted in an instant and permanent threshold shift of 96 dB in the affected ear. A massive reduction of cochlear nerve fibers in the ventral cochlear nucleus (VCN) was demonstrated by tracing them from the cochlea of rats that survived acoustic overstimulation for 1 year or longer. In the auditory brainstem, we observed a deprivation-dependent appearance of fibers positive for tyrosine receptor kinase B in the ipsilateral VCN between day 3 and day 21 after trauma and an increase in phosphoserine immunostaining in the neuropil of the ipsilateral VCN and in neurons of the contralateral lateral superior olive during the first 30 days after trauma. Immunoreactivity for the cAMP response element binding protein in its phosphorylated form was transiently depressed in the ipsilateral inferior colliculus immediately after trauma and was elevated as late as 7 months after trauma in the ipsilateral VCN. Apparently, a unilateral acoustic overstimulation entails specific regulations of the activity of plasticity-associated molecules through phosphorylation and includes changes to neurotrophin signaling between neurons of the auditory brainstem.

We explored the consequences of unilateral acoustic trauma to intracochlear and central nervous system structures in rats. An acoustic trauma, induced by applying click stimuli of 130 dB (sound pressure level; SPL) for 30 minutes, resulted in an instant and permanent threshold shift of 95.92 ± 1.08 dB (SEM) in the affected ear. We observed, as a consequence, a structural deterioration of the organ of Corti. Deprivation‐dependent changes of neurons of the auditory brainstem were determined using antibodies against neurofilament and the growth‐associated protein GAP‐43 and compared with those following cochleotomy, studied earlier. By 231 days posttrauma, spiral ganglion cell bodies and their processes were almost entirely lost from all cochlear regions with destroyed organ of Corti. In the lateral superior olive (LSO) ipsilateral to the trauma, cell bodies of lateral olivocochlear neurons turned transiently GAP‐43 positive within the first 1.5 years posttrauma. The time course of emergence and disappearance of this population of neurons was similar to that found after cochleotomy. Additionally, after noise trauma, principal cells in contralateral LSO and in medial superior olive (MSO) on both sides of the brainstem developed an expression of GAP‐43 that began 3 and 16 days posttrauma, respectively, and lasted for at least 1 year. Such cells were rarely observed after cochleotomy. An unequivocal rise in GAP‐43 immunoreactivity was also found in the neuropil of the inferior colliculus and the ventral cochlear nucleus, both preferentially on the acoustically damaged side. We conclude that the degree and specific cause of sudden unilateral deafness entail specific patterns of plasticity responses in the auditory brainstem, possibly to prevent the neural network dedicated to locate sounds in the environment from delivering erroneous signals centralward. J. Comp. Neurol. 451:250–266, 2002. © 2002 Wiley‐Liss, Inc.

Neuronal activity in sensory organs elicited by adequate or electrical stimulation not only invokes fast electrical responses but may also trigger complex molecular changes inside central neurons. Following electrical intracochlear stimulation with a cochlear implant under urethane anesthesia, we observed changes in the phosphorylation state of the cAMP response element binding protein (CREB) and the expression of the immediate-early genes c-fos and egr-1, molecules known to act as transcription factors, in a tonotopically precise pattern in central auditory neurons. These neurons resided in the posteroventral and anteroventral cochlear nucleus, the dorsal cochlear nucleus, the lateral superior olive, the medial nucleus of the trapezoid body, the dorsal and ventral nucleus of the lateral lemniscus, and the central nucleus of the inferior colliculus. Moreover, effects of electrical stimulation were identified in the medial vestibular nucleus and the lateral parabrachial nucleus. Regionally, CREB was dephosphorylated wherever immediate-early gene expression went up. These massive stimulation-dependent modulations of transcription factors in the ascending auditory system are indicative of ongoing changes that modify the chemistry and structure of the affected cells and, consequently, their response characteristics to subsequent stimulation of the inner ear.

Investigating activity-dependent plasticity in the auditory brain stem of the adult rat, we observed that electrical intracochlear stimulation led to a tonotopically localized modulation of the phosphorylation of the cAMP response element binding protein (CREB) and an equally localized expression of the immediate early gene product c-Fos in cochlear nucleus and superior olive. As P-CREB is thought to act as transcription factor on one promoter site of the c-fos gene, we compared immunolabeling for P-CREB and c-fos in adjacent brain sections. Follow ing 2 h sustained stimulation in previously deafened animals, labelling for P-CREB declined in regions where c-Fos labelling increased. This suggests that the level or state of P-CREB (e.g. whether it is phosphorylated or not) are affected by intracochlear stimulation in a process that appears to be linked to the stimulation-dependent expression of c-Fos in auditory brain stem nuclei.