Учебники / Auditory Trauma, Protection, and Repair Fay 2008

Figure 9.2. Guinea pig ventral CN after unilateral cochlear ablation. (A) In the AVCN on the ablated side, synaptophysin-containing clusters in the neuropil (Clusters) and those surrounding cell bodies (Pericellular) declined by 4 days after ablation, consistent with the loss of cochlear nerve endings. Resurgence of these profiles at 7 days is consistent with a growth of new synaptic endings. Asterisks denote a difference from the unlesioned control; bars containing lower case letters differ from those with the same letter below (paired t-test). Data from Benson et al. (1997). (B) BDNF was elevated first, at 3 days, in the ventral CN on the ablated side (IPsi), while NT3 became elevated later, at 7 days. These changes may have contributed to the survival of deafferented CN neurons and to the synaptogenesis illustrated in A. On the intact side (Contra), BDNF deficiencies appeared at 3 days and recovered by 7 days, while NT3 levels became elevated by 7 days. (C) At 5 days after ablation, during the period of synaptogenesis (A), and when neurotrophins were elevated (B), ERK1/2-P levels increased in AVCN neurons on the ablated side, and ERK1/2-P was transported mainly into the cell nucleus. Scale bar = 25 μm. (D) Quantification of bands in Western blots confirmed the elevation of ERK2-P levels at 3 and 7 days after ablation. Asterisks denote a difference from the unlesioned control (Mann–Whitney test). These findings (in C and D) may reflect a relationship between increased neurotrophic support and altered gene expression, via increased ERK cell signaling activity, which contributed to the growth of new synaptic endings in the AVCN during the first week after ablation. (C and D from Suneja and Potashner 2003.)

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changes and some of the pathological signs (Jastreboff 1990; Kaltenbach and Afman 2000; Salvi et al. 2000; Brozoski et al. 2002) are consistent with the view that such alterations generate or contribute significantly to the symptoms in sensorineural hearing loss.

The aforementioned examples of plasticity can be viewed as altered cellular phenotypic behaviors that probably originate from molecular signals that appear when the cochlear nerve is damaged or lost. The earliest changes might be manifested in the CN and could include the loss of transmission and trophic support provided by the cochlear nerve endings. Also, signals might include biochemical factors released from the degenerating cochlear nerve and its endings or induced in other cells by cochlear nerve loss. The signals presumably generate successive cascades of cellular responses (via signal transduction pathways) and subsequent biochemical signals, followed by further cellular responses and signals. The cascades would emanate from the CN, travel through the auditory pathway to other auditory nuclei, and shape the pathobiology of the hearing loss. The development and course of CN alterations may initiate and sustain plastic changes in higher auditory nuclei. Similarly, plastic changes in the brain stem nuclei might contribute to the development of changes in the thalamus and cortex, all of which might feed back to the CN. Kaltenbach et al. (2005) suggest that the dorsal CN is the primary site for the origin of central hyperactivity after noise exposure. This does not necessarily exclude a contribution from other regions, especially the ventral CN (see also Bauer and Brozoski, Chapter 4). For it is hard to believe that the extensive remodeling described in the ventral CN has no consequences for hearing.

Our concept of the critical events for sensorineural hearing loss is supported by the previous findings, some of which indicate that neurotrophins, upregulated after sensorineural hearing loss, probably function as relatively early and long-term signals (Suneja et al. 2004). Preliminary data suggest that additional signals may consist of other cytokines (Suneja and Potashner, unpublished). However, the complement of plasticity-inducing molecules and the cells that produce them remain to be identified. Despite this gap in our knowledge, evidence suggests that such signals do act to change the behavior of central auditory cells and the function of central auditory pathways. Signals, such as cytokine and neurotrophin proteins, typically bind to and activate cell surface receptors, which in turn activate one or more signal transduction pathways. In effect, the signal transduction pathways transduce the initial set of signals to produce altered cell morphology and behavior by modifying a variety of cellular proteins, eliciting regulatory changes in cellular pathways and altering gene expression.

The activity of such a process in the central auditory nuclei is evident after sensorineural hearing loss. For example, cochlear ablation altered activity in the ERK (extracellular regulated kinase) signal transduction pathway (Fig. 9.2C,D) (Suneja and Potashner 2003), suggesting that such pathways respond to signals that appear after the lesion. In addition, cochlear ablation altered the activation of CREB (Mo et al. 2006), a protein that controls gene expression, and changed the

activation of factors that control protein synthesis (Mo et al. 2006). Since these changes were roughly coincident with the post-ablation growth of new ectopic synapses mentioned previously (Benson et al. 1997; Muly et al. 2004), they suggest that signal transduction activity was responsible for these morphological rearrangements.

Cochlear ablation also brought about altered transmitter release and synaptic receptor activities in central auditory nuclei that were consistent with a loss of inhibitory glycinergic synaptic activity and an increase in excitatory glutamatergic activity (Potashner et al. 1997, 2000; Suneja et al. 1998a,b, 2000; Suneja and Potashner 2003). Moreover, many of these plasticities depended on protein kinases (Zhang et al. 2002, 2003a,b, 2004; Yan et al. 2006) that were themselves sensitive to changes in signal transduction activity. It is likely, therefore, that the regulation of synaptic transmission in central auditory nuclei is governed by signal transduction mechanisms.

3. Effects of Acoustic Overstimulation

Structural and functional reorganization may occur in the adult central nervous system in response to direct or indirect perturbations, including sensory deprivation and overstimulation (Johnson 1975; Merzenich et al. 1983; Erb and Povlishock 1991; Diamond et al. 1993; Rajan et al. 1993). At the level of the spinal cord, damage of peripheral sensory nerves in adult animals may elicit structural reorganization in the gray matter, with the sprouting of new axon terminations (Goldberger and Murray 1985; LaMotte and Kapadia 1993). In the auditory system, exposure of adult animals to intense sound can produce cochlear lesions and hearing impairment (Bohne 1976). The location of the damage in the cochlea reflects the spectral composition of the traumatizing sound (Eldredge et al. 1981). The noise-induced hearing loss may be associated with abnormal auditory functions, such as loudness recruitment or tinnitus (Axelsson and Barrenas 1992; Jastreboff and Jastreboff 2002), and with degenerative changes in the central auditory pathways (Saunders et al. 1991). Animal models have implicated prolonged hyperactivity and decrease of inhibitory transmitters in tinnitus (e.g., Kaltenbach and McCaslin 1996; Milbrandt et al. 2000; Salvi et al. 1992; Chang et al. 2002).

3.1 Fiber Degeneration and Evidence of a New Growth of Axons

Following acoustic trauma, the patterns of terminal degeneration in the CN are similar to those after ablation, but they have several differences (Kim et al. 1997). Heavy terminal degeneration still collects in bands that correspond to the tonotopic locations of the inner hair cell and myelinated fiber lesions (Fig. 9.3). However, unlike ablation, the regions between bands also contain degenerated axons, thinner and in lower numbers. Terminal degeneration can be

but not in the expected cochleotopic pattern, and unexpected zones of terminal degeneration appear.

In the early studies, cats survived for up to 3 years after acoustic damage to the middle turn of the cochlea (Morest et al. 1979, 1987; Morest 1982; Morest et al. unpublished findings). The large cochlear nerve endings, including the endbulbs of Held in the ventral CN, disappeared gradually over a period of months. After 3 years, the coarser fiber plexus associated with cochlear nerve endings was gone. Instead there was a marked infiltration of a plexus of very fine axons, clearly visible in silver impregnations, throughout the ventral CN. This appeared to be a new growth of axons. In electron microscopic reconstructions of bushy cell bodies, following mechanical ablation of the cochlea, the largest synaptic endings with the primary-like cytology of cochlear nerve endings disappeared. A similar result followed acoustic trauma, except that there was also a large shift in the ratio of axosomatic endings to the smaller terminals, mostly without primary-like cytology.

In summary, the evidence suggests that acoustic trauma can initiate a structural reorganization of synaptic connections in the cochlear nucleus and central auditory pathways. Such a reorganization might entail axonal pruning, dendritic remodeling, and the growth of new axons and synapses. A disturbance in the ratio of excitatory to inhibitory endings provides a potential basis for explaining hyperactivity and tinnitus. Terminal degeneration continued for at least 8 months, suggesting that this may be a progressive disease.

3.2 A New Growth of Axons and Synapses

The anterior part of the posteroventral CN, dorsal subdivision (PVCNA), is a good place to begin a detailed study of the noise-induced changes, because it contains all of the major neuronal types found in the ventral CN, thus providing a veritable microcosm of the auditory brain stem. After acoustic trauma in the chinchilla, PVCNA consistently contained heavy terminal degeneration, followed by the reappearance of many normal axons, as first shown by Bilak et al. (1997). Comparisons of these axon concentrations to those in equivalent areas of unexposed and protected controls indicates that this deafferented zone initially lost half of its axons, followed by a partial recovery of small diameter axons (Kim et al. 2004a,b,c) (Fig. 9.4). During this recovery, the number of axonal endings on neuronal cell bodies increased. These findings imply that changes in the PVCNA initiated by acoustic trauma include initial deafferentation, followed by the sprouting of new, small diameter axons and the formation of new synaptic contacts. There was a marked increase in the ratio of excitatory to inhibitory synapses. Terminal degeneration and new growth of axons continued for at least 8 months, suggesting that this may be a progressive disease. The result is a reorganization which may well contribute to hyperexcitability and increased spontaneous activity and lead to tinnitus and loudness misperceptions.

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Figure 9.4. Normal endings after noise exposure in chinchillas. (After Kim et al. 2004c). (A) Excitatory (large+small round vesicles) compared to (B) inhibitory (pleomorphic + flat vesicles) for unexposed controls (Con) and exposed. Bars = mean + SEM as % of total number in controls. (C) Comparison of combined sums from A and B: KolmogorovSmirnov, p < 0.05.

4. Key Molecules Underlying the Synaptic Reorganization

Following Noise

There is little information available on the molecular basis for the pathological changes in the brain due to noise damage. Only a few reports address the changes following cochlear ablation or aging with respect to neurotransmitters and growth factors.

Studies of the transmitter-related receptor molecules have been reviewed by Sato et al. (2002). In the brain, the chief inhibitory transmitters are-aminobutyric acid (GABA) and glycine, whereas glutamate is generally excitatory. The effect of a transmitter depends on activation of its specific receptor. In the case of glutamate there are many different kinds of receptors with different properties, including ionotropic( -amino-3-hydroxy-5- methylisoxazole-4-propionic acid [AMPA] and N-methyl-d-aspartate [NMDA]) and metabotropic (not directly linked to ion channels). In brief, the literature suggests that there may be significant changes in expression of GABA, glycine, AMPA, and NMDA receptors. In their own experiments following cochlear ablation, those authors report decreases of these receptor mRNAs in certain cells in the CN of rats by using in situ hybridization after 5 days, but there was a complete restoration by 20 days. In comparison, Potashner et al. (1997, 2000) and Suneja et al. (1998a,b), using biochemical assays in vitro, have documented an increase in glutamate release as well as a decline in glycine release and glycine receptor binding shortly after cochlear ablation in guinea pigs. Other receptors and channels are of interest, including metabotropic receptors, sodium, potassium, and calcium channels.

After noise exposure in chinchillas, Muly et al. (2004) reported an early increase in glutamatergic release and decreased uptake in the first few days in the CN. By 14 days, there was a decrease in glutamatergic uptake and release. Finally, after 90 days, there was a recovery of release and an increase in AMPA receptor binding. These findings are consistent with an early excitotoxic disturbance, followed by a dystrophic disorder. The subsequent recovery of glutamate activity and overexpression of the receptor are consistent with a dystrophic effect. Later increases in glutamatergic release and AMPA receptor activity are consistent with the new growth of axons and their synapses.

Growth factors are specialized proteins known to promote development of neurons and their connections. For example, fibroblast growth factors (FGF) have a widespread distribution in the nervous system and act over broad time periods in development. Neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) play specific roles in the differentiation of neurons and their synapses. Specific receptors for these factors have been identified. These same factors and receptors may become active in the pathological responses of the nervous system.

Some studies address the role of growth factors involved in damage to the cochlea, but few look at the effects in the CN. Studies of the role of FGFs in the development of the auditory system (e.g., Hossain et al. 1995, 1997; Zhou et al. 1996) suggest that FGF1 and FGF2 and their receptors are critical in switching spiral ganglion cells and their target cells in the CN from a proliferation mode to one favoring migration and process outgrowth (Brumwell et al. 2000; Hossain and Morest 2000; Hossain et al. 2002; Bilak et al. 2003). FGF2 can act by upregulating the TrkB receptor, which is the high-affinity receptor for BDNF, whereupon process outgrowth and targeting of sensory and central neurons are accelerated. Finally NT3, together with FGF2 and BDNF, upregulate the TrkC receptor, which is the high-affinity receptor for NT3. At that stage, axonal maturation and target selection, together with synapse formation, appear. The details differ in the chicken and mouse (Brumwell et al. 2005; Hossain et al. 2006). In the adult mouse, expression of these molecules may be upregulated in response to noise damage (Smith et al. 2002). The rationale is that, after the excitotoxic and dystrophic events which result from overstimulation, the genes for these molecules are upregulated in response to the renewed opportunity for axonal growth and new synapse formation provided by the loss of synaptic endings from the central neurons. Some support for this rationale comes from studies of cochlear ablation, where the first week after ablation was marked by elevations of BDNF in the ventral CN, followed by increases of NT3 (Fig. 9.2B) and a growth of new synapses (Fig. 9.2A) (Suneja et al. 2005).

5. Summary and Conclusions

Cochlear damage in adult animals induces changes in central auditory neurons and glia, such as degeneration of axons and dendrites, followed by growth of new axonal endings, synaptic reorganization, and altered transmitter biochemistry and physiology. These plasticities are thought to underlie the pathology and

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symptoms. For example, a disturbance in the ratio of excitatory to inhibitory endings in the cochlear nucleus provides a potential basis for explaining central hyperactivity and tinnitus. Also the long-term continuation of synaptic degeneration in the brain after noise damage suggests that noise-induced hearing loss may be a progressive disease.

In the research on sensorineural hearing loss in animal models, three main concepts have emerged. First, there is strong evidence that noise-induced hearing loss resulted in initial synaptic depletion, followed by synaptogenesis and synaptic rearrangements in the cochlear nucleus. This creates a net loss of inhibitory synaptic contacts and the establishment of ectopic terminals, many of which exhibit the morphology of excitatory synapses. These events provide a structural basis for the well known hyperexcitability of the auditory pathway and its deficient inhibition after sensorineural hearing loss. The findings may also indicate a structural basis for the signs and symptoms that accompany sensorineural hearing loss, such as tinnitus and loudness misperceptions.

Second, activity in the signal transduction pathways may be altered after unilateral cochlear ablation, implying that signals and signal transduction may play a role in altering gene expression, endogenous regulation, and phenotypic cell behavior in auditory nuclei with sensorineural hearing loss. Temporal correlations between the expression of signaling molecules and/or signal transduction events, on the one hand, and synaptogenesis and/or changes in synaptic biochemical activities, on the other hand, suggest that altered signal transduction activity might control synaptic reorganization. This implies that after sensorineural hearing loss, synaptic plasticity, and thus pathological changes, might be alleviated by manipulations of signal molecules and signal transduction activity.

Third, neurotrophins and cytokines have emerged as candidate signal molecules that might generate the central, plastic changes in signal transduction activity after sensorineural hearing loss. These plasticities may be correlated with altered expression of neurotrophins, growth factors, neurotransmitters and their receptors, and altered activity in signaling molecules, such as protein kinases and the cyclic-AMP response element binding protein. These discoveries allow us, for the first time in the auditory field, to study the cellular and molecular mechanisms of sensorineural hearing loss in the brain as well as the cochlea.

Acknowledgments. The authors’ research is supported by NIH grants DC000127 (D.K.M.) and DC000199 (S.J.P.).

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