The dorsal cochlear nucleus as a contributor to tinnitus: mechanisms underlying the induction of hyperactivity

B. Langguth, G. Hajak, T. Kleinjung, A. Cacace & A.R. Møller (Eds.) Progress in Brain Research, Vol. 166 ISSN 0079-6123 Copyright r 2007 Elsevier B.V. All rights reserved

CHAPTER 9

The dorsal cochlear nucleus as a contributor to tinnitus: mechanisms underlying the induction of hyperactivity James A. Kaltenbach Department of Otolaryngology, Head and Neck Surgery, Wayne State University School of Medicine, Detroit, MI 48201, USA

Abstract: It has been hypothesized that tinnitus percepts may arise, in part, from increases in spontaneous neural activity in the central auditory system. The DCN is the lowest central auditory nucleus where this hyperactivity is observed, and it is most prominent following exposure to intense sound or ototoxic insult. Efforts to develop effective treatments for tinnitus will probably benefit from a better understanding of the mechanisms underlying the induction of hyperactivity in the DCN. This chapter will summarize the evidence linking tinnitus to altered activity in the DCN and review some of the likely mechanisms underlying the induction of hyperactivity following injury to the ear. Keywords: tinnitus mechanisms; plasticity; dorsal cochlear nucleus; disinhibition; hyperactivity The evidence comes from studies using a variety of functional measures, such as single- and multi-unit recordings, c-fos immunocytochemistry and 2-deoxyglucose metabolic mapping (Jastreboff and Sasaki, 1986; Kaltenbach and McCaslin, 1996; Manabe et al., 1997; Eggermont and Kenmochi, 1998; Kaltenbach et al., 1998, 2002; Kimura and Eggermont, 1999; Kaltenbach and Afman, 2000; Komiya and Eggermont, 2000; Norena and Eggermont, 2003; Wallhausser-Franke et al., 2003; Ma et al., 2006). These studies have shown that the same tinnitus-inducing agents that cause animals to experience tinnitus also cause increases in spontaneous activity. This hyperactivity has been observed at several levels of the central auditory system, including the dorsal cochlear nucleus (DCN), the inferior colliculus (IC) and the auditory cortex (AC). The hypothesis that increased spontaneous activity represents a neural

Introduction Over the past decade, an increasing number of animal models have been developed for the study of tinnitus mechanisms. Studies using these models have consistently demonstrated that agents which cause tinnitus in humans, such as sodium salicylate, noise and quinine, cause animals to experience tinnitus-like percepts (Jastreboff et al., 1988; Bauer et al., 1999; Brozoski et al., 2002; Heffner and Harrington, 2002; Guitton et al., 2003; Lobarinas et al., 2004). Although many changes in neuronal function have been observed in these animal models, the most frequently reported alteration is the condition of hyperactivity, characterized as an increase in spontaneous activity. Corresponding author. Tel.: +1 313 577 1257; Fax: +1 313 577 0085; E-mail: [email protected] DOI: 10.1016/S0079-6123(07)66009-9

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correlate of tinnitus has gained further strength from imaging studies showing hyperactivation of auditory areas in the brains of human subjects with tinnitus (Shulman, 1995; Arnold et al., 1996; Lockwood et al., 1998; Giraud et al., 1999; Andersson et al., 2000; Melcher et al., 2000; Mirz et al., 2000; Wang et al., 2000; Lockwood et al., 2001). The DCN is the lowest level in the central auditory system where tinnitus-related hyperactivity has been observed (see reviews of Eggermont and Roberts, 2004; Kaltenbach et al., 2005). This structure has been implicated in localization of sounds in three-dimensional space, particularly in the vertical plane (Masterton et al., 1994; May, 2000); however, its connections with numerous other structures, both auditory and non-auditory, suggest that the DCN has additional functions (see Kaltenbach, 2006). Its involvement in tinnitus is of special interest for several reasons. First, because the DCN receives direct innervation from the auditory nerve, it is especially vulnerable to alterations of peripheral input, such as those that accompany cochlear injury resulting from exposure to tinnitus-inducing agents. Second, because its output is relayed to higher order auditory centers, it is in a position to influence, and perhaps even contribute to increases in, the levels of activity higher up in the auditory pathway. Third, it is a center of integration of different sensory modalities, and as such, provides cross-modal interactions that could offer avenues for treatment beyond the borders of the auditory system. The DCN as a contributor to tinnitus Numerous lines of evidence support the hypothesis that the DCN is an important contributor to tinnitus percepts. This evidence has recently been reviewed (Kaltenbach, 2006) and is summarized briefly here: (1) Direct electrical stimulation of the DCN has been reported to cause changes in the loudness of tinnitus (Soussi and Otto, 1994). The evidence comes from a study of 10 tinnitus patients with neurofibromatosis-2 (NF-2), each of whom received an auditory

brainstem implant (ABI) following bilateral acoustic neuroma (vestibular Schwannoma) surgery. In each patient, the ABI was implanted unilaterally on the surface of the DCN. The patients were asked to describe how their tinnitus percepts were affected during periods of ABI stimulation. Seven of the 10 patients reported a decrease in the loudness of their tinnitus during ABI stimulation, one reported an increase and two reported no change in their tinnitus. Some patients also reported changes in the pitch of their tinnitus or in the number of tinnitus sounds. However, the changes in tinnitus perception occurred only as long as the period of stimulation; there was no evidence of residual inhibition. The results of this study, though preliminary in nature, suggest that changes in tinnitus percepts are related to changes in the level of neural activity in the DCN. Since changes in tinnitus can also be elicited by stimulating the cochlea, the findings with the ABI stimulation may indicate that the DCN is a key part of a tinnitus-generating circuit that can be influenced by changes in the level of input from the auditory periphery. (2) Agents that cause tinnitus in humans also cause increases in spontaneous neural activity in the DCN of animals. This condition of hyperactivity, observed at both multi(Fig. 1) and single-unit levels, occurs following either noise or ototoxic drug exposure, and has been demonstrated in several species, including hamsters (Kaltenbach and McCaslin, 1996; Kaltenbach et al., 1998, 2000, 2002, 2005), rats (Zhang and Kaltenbach, 1998), guinea pigs (Imig and Durham, 2005), mice (Kaltenbach et al., 2001), chinchillas (Brozoski et al., 2002) and gerbils (Wallhausser-Franke et al., 2003). This suggests that noise-induced hyperactivity in the DCN is an across-species phenomenon, although it has not yet been reproduced in brain slices (Chang et al., 2002) or in decerebrate animals (Ma and Young, 2006). (3) The hyperactivity induced in the DCN by intense sound exposure resembles the

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Fig. 1. Comparison of the levels of DCN spontaneous activity recorded in two groups of animals, one exposed to an intense (127 dB SPL) 10 kHz tone for 4 h (gray bars), the other serving as unexposed control animals (black bars). Mean activity is plotted for each group as a function of distance along the medial-lateral (tonotopic) axis of the DCN. Points along the X-axis represent distances from the lateral part of the DCN. The data show consistently higher levels of mean spontaneous activity (hyperactivity) in exposed animals relative to those of controls.

increases in activity evoked in the DCN of normal animals during moderate sound stimulation. For example, the distribution of hyperactivity across the tonotopic range displays a profile with a distinct peak. This profile is similar to what is observed in the DCN during stimulation with a moderate level 10 kHz tone (Kaltenbach and Afman, 2000). This similarity suggests that noiseinduced hyperactivity in the DCN carries a place code for a tonal stimulus, which corresponds to the typical tonal character of tinnitus. (4) DCN hyperactivity displays several features that are in line with the psychoacoustic features of tinnitus. For example, although tinnitus usually has a distinct pitch, pitch matching studies have shown that tinnitus has a spectral profile more like that of a narrow band of noise (Norena et al., 2002). Similarly, the tonotopic spectrum of DCN hyperactivity is more like the profile of activity evoked by a narrow band of noise than by a pure tone (Kaltenbach and Afman, 2000). Moreover, the peak of the spontaneous activity profile occurs at a tonotopic

locus representing a frequency that is higher than that of the inducing exposure tone (Kaltenbach and Afman, 2000). This agrees with the psychophysical finding that noiseinduced tinnitus is typically matched to a frequency that is higher than that of the exposure tone (Loeb and Smith, 1967; Atherley et al., 1968). (5) Noise-induced hyperactivity in the DCN is correlated with tinnitus. This has been shown by behavioral studies demonstrating that animals exposed to the same intense sound conditions that cause hyperactivity in the DCN develop tinnitus-like percepts (Brozoski et al., 2002; Heffner and Harrington, 2002). Moreover, when spontaneous activity was recorded in the same animals that had previously been tested behaviorally for tinnitus, the behavioral measures of tinnitus were found to have a statistically significant correlation with the peak level of activity in the DCN (Kaltenbach et al., 2004). (6) The DCN possesses the type of circuit connections with the somatosensory system needed to explain some forms of somatic

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tinnitus. For example, tinnitus can sometimes be modulated by stimulating the median nerve. In 80% of tinnitus patients, the loudness or pitch of their tinnitus can be modulated by certain manipulations of the head and neck musculature (Møller et al., 1992; Levine, 1999; Møller and Rollins, 2002). This is particularly noticeable when muscles innervated by the trigeminal and upper cervical nerves, especially C2 and C3 are contracted (Levine, 1999, 2004; Levine et al., 2003; Abel and Levine, 2004). An important feature of this form of somatic tinnitus is that, when unilateral, the effective manipulation involves muscles on the side ipsilateral to the tinnitus. In addition, when accompanied by other craniofacial pathologies, the pathologies are usually on the side ipsilateral to the tinnitus (Levine, 2004). This would seem to require a circuit in which auditory inputs are integrated with ipsilateral somatosensory inputs. The DCN is one such structure, and it may be the only one where this integration is predominantly ipsilateral (Itoh et al., 1987; Weinberg and Rustioni, 1987; Wright and Ryugo, 1996; Shore et al., 2000; Shore, 2004, 2005; Zhao and Shore, 2004). Moreover, electrophysiological studies have shown that spontaneous activity of DCN neurons can be modulated by stimulation of the cervical nerves (especially C2) or certain ipsilateral cranial nerves, including the sensory branch of the trigeminal nerve (Kanold and Young, 2001) or the trigeminal ganglion (Shore, 2005). (7) The DCN exhibits several forms of neuronal plasticity that parallel the various forms of plasticity that characterize tinnitus. These have recently been reviewed (Kaltenbach et al., 2005). For example, both tinnitus and DCN hyperactivity often develop as a consequence of cochlear outer hair cell injury (Melamed et al., 2000; Kaltenbach et al., 2002; Rachel et al., 2002), and both persist following eighth nerve destruction (Zacharek et al., 2002). Another example of tinnitus plasticity is its common tendency to change in loudness and pitch over time (Penner,

1983, 1995; Tyler and Conrad-Armes, 1983, 1984; Burns, 1984; Meikle, 1987; Penner and Bilger, 1992). Similarly, changes occur in the magnitude and tonotopic locus of DCN hyperactivity over time (Kaltenbach et al., 2000). (8) The DCN possesses circuitry and physiological properties that could explain gazeevoked tinnitus. This form of tinnitus sometimes develops following surgeries that result in injury to the eighth nerve, and is characterized by a modulation of tinnitus that occurs when the angle of gaze is changed (Wall et al., 1987; Cacace et al., 1994a, b). A possible basis for this modulation may be the input that the DCN receives indirectly from Roller’s nucleus, a vestibular-related nucleus (Kaufman et al., 2000), which is part of the brainstem perihypoglossal complex; this complex is believed to be involved in the coordination of eye movements during head displacements (McCrea et al., 1987). Neurons in Roller’s nucleus project to the granule cell domain of the CN (Ryugo et al., 2003), which modulates the activity of DCN neurons. Input from this nucleus could underlie changes in the level of multiunit activity in the DCN that occur during changes in eye position during slow-wave sleep (Mori et al., 1972). Gaze-evoked tinnitus could be explained as a condition in which DCN neurons become sensitized to input from the Roller’s nucleus-granule cell pathway. Such changes might be triggered by damage to the eighth nerve.

The importance of higher auditory centers in tinnitus There is also evidence for the involvement of the IC and AC in tinnitus. Studies conducted in mice, using electrophysiological recording methods, have shown evidence for increased spontaneous activity in the central nucleus of the inferior colliculus (ICC) following intense noise exposure (Ma et al., 2006). Similar increases in spontaneous activity have been reported for this nucleus in

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guinea pigs treated with sodium salicylate (Jastreboff et al., 1986; Chen and Jastreboff, 1995; Manabe et al., 1997). Studies in human subjects using fMRI have also shown that some forms of tinnitus are associated with elevated activity in the IC (Melcher et al., 2000). At the cortical level, activity increases have been demonstrated electrophysiologically in the primary AC of anesthetized cats following noise exposure (Kimura and Eggermont, 1999; Komiya and Eggermont, 2000) and in the secondary AC following treatment with salicylate or quinine (Ochi and Eggermont, 1997; Eggermont and Kenmochi, 1998). Numerous studies using positron emission tomography (PET) have shown evidence of increased activation of auditory cortical areas in human subjects with tinnitus (Arnold et al., 1996; Lockwood et al., 1998; Giraud et al., 1999; Andersson et al., 2000; Mirz et al., 2000; Wang et al., 2000; Lockwood et al., 2001). The increased activation of AC of patients with tinnitus can be modulated in the upward or downward direction by administration of lidocaine (Andersson et al., 2000; Reyes et al., 2002). In individuals whose tinnitus was made louder by lidocaine, the AC showed an increase in the level of activation in the AC, whereas those experiencing a decrease in loudness had a corresponding decrease in cortical activation (Reyes et al., 2002). However, the role of AC in tinnitus perception may be more complex than previous studies might suggest. Arnold et al. (1996) observed increased activation in the primary AC in patients who were chronically troubled by their tinnitus, but not in a chronic tinnitus patient who had no subjective complaints. They also observed increased activation of the primary AC in a chronic tinnitus patient during periods when the tinnitus was disabling, but not during periods when the patient was experiencing relief. This could mean that the AC displays increases in activity only when the tinnitus reaches some critical threshold of severity. This possibility suggests that activity in the AC might be correlated with a negative emotional response to tinnitus (see Chapter 20). Alternatively, the AC might be involved in directing attention to tinnitus, which may tend to occur when the tinnitus is more severe or troubling.

Mechanisms underlying the emergence of hyperactivity in the DCN The changes leading to hyperactivity in the DCN may involve many different mechanisms. For example, increases in activity after noise exposure might result from shifts in the balance of excitation and inhibition at the synaptic level (Fig. 2). These shifts could arise as a direct consequence of injury or degeneration, produced either by transneuronal cell loss (Fig. 2A) or by excitotoxicity injury (Fig. 2B). In both cases, loss of normal input might trigger plastic adjustments at the synaptic level that affect the release of neurotransmitters, the number of postsynaptic receptors or even the number of synapses further downstream. Plasticity (see Chapter 3) could also lead to changes in intracellular signaling pathways that control the expression of various ion channels, thus altering the intrinsic membrane properties of neurons (Fig. 2C). This section will review literature, describing examples of each type of change, and discuss evidence for their possible involvement in the generation of neural hyperactivity and tinnitus.

Neural degeneration as a possible basis of hyperactivity Neural degeneration in the adult central auditory system is a potentially important mechanism by which injury to the inner ear can cause hyperactivity in the DCN, since it directly affects the relative strengths of excitatory and inhibitory inputs to DCN neurons (Fig. 2). A greater loss of inhibitory synapses might cause a net disinhibition of neurons that increases their levels of spontaneous activity. The type and distribution of degenerating synapses in the CN following manipulation that are known to cause tinnitus will be discussed below. Morest and Bohne (1983) and Kim et al. (1997) reported widespread degeneration of thick- and thin-fiber populations in all subdivisions of the chinchilla CN following octave band noise exposure. Degeneration first became apparent 4–8 days after exposure. More detailed studies of degeneration in the CN following intense noise exposure

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Fig. 2. Three possible mechanisms by which intense sound exposure causes an increase in spontaneous activity of DCN neurons. The first mechanism (Path A) shows increases in spontaneous activity to be the result of changes in the balance of excitatory and inhibitory inputs to DCN neurons resulting from transneuronal degeneration and plasticity triggered by cochlear injury. The second mechanism (Path B) shows a similar shift in the balance of excitatory and inhibitory inputs to DCN neurons; however, in this case the shift is due to excitotoxic injury of DCN neurons resulting from overstimulation and hyperactivity of the auditory nerve and/or excitatory neurons in the DCN. The third mechanism (Path C) shows increased activity to be the product of the intrinsic membrane properties of neurons. Such changes are brought about by altered expression of ion conductance channels that affect the cells’ level of excitability. The changes in ion channel expression results from plasticity that is triggered either by hyperactivition of the auditory nerve or by loss of normal input caused by cochlear injury.

have revealed several important features. First, hair cell loss associated with degeneration of primary afferent dendrites leads to loss of fibers in the CN; in each subdivision of the cochlear nucleus, the fiber loss was found to be concentrated in one or more bands corresponding to the tonotopic loc(i)us of damaged or missing inner hair cells and/ or degenerated myelinated nerve fibers observed in the cochlea (Kim et al., 1997). Some of this degeneration was limited to loss of primary afferent axons, but there was also evidence for transneuronal degeneration (see also Sie and Rubel, 1992; Zhao and Lurie, 2004). For example, after loss of eighth nerve fibers, bands of degeneration have been observed in other brainstem structures, such as the superior olivary complex (SOC) and ICC with tonotopic loci similar to those in the CN, even though no cochlear nerve fibers project further centrally than the CN (Morest et al., 1997). [Note: There is some evidence that noise exposure can also induce cell death in the DCN,

anteroventral cochlear nucleus (AVCN) and SOC by apoptosis, although the apoptotic cell types have not yet been defined (Aarnisalo et al., 2000)]. Second, degeneration sometimes appears in areas of the CN, especially the DCN, without any corresponding loss of inner hair cells or primary afferent dendrites; this degeneration was interpreted as possibly being due to excitotoxicity (Kim et al., 1997). Third, the degeneration process can continue for many months. The numbers of degenerated fibers and degenerated pre-synaptic terminals are not constant but vary considerably over time; these degenerative changes are accompanied by sprouting of axons and formation of new synapses (Benson et al., 1997; Bilak et al., 1997; Kim et al., 2004a, b). Thus, noise-induced injury is a chronic, ongoing problem that continues long after termination of the exposure and beyond the period of primary afferent degeneration. Fourth, the degeneration and re-growth processes result in a greater loss of inhibitory than

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excitatory synapses (Kim et al., 2004b). This is because excitatory synapses in the ventral cochlear nucleus (VCN) make a more complete recovery than inhibitory synapses. This recovery may thus favor an increase in excitation. An increase in excitation is consistent with the losses of glycinergic neurotransmission in the VCN following cochlear ablation (Suneja et al., 1998a, see below). A similar relationship may exist in the DCN where noise exposure causes increases in spontaneous activity and decreases in glycinergic input (Asako et al., 2005).

Changes in cell size as a possible basis of hyperactivity Cell shrinkage is another variant form of degeneration. Reductions in the size of cochlear nucleus cells have been observed following various kinds of manipulations that affect primary afferent input to the CN, such as blockage of outer or middle ear conduction (Webster and Webster, 1979), pharmacological block of primary afferent activity (Pasic and Rubel, 1989), induction of ototoxic injury (Lustig et al., 1994; Lesperance et al., 1995; Kawano et al., 1997) and anatomical deafferentation by cochlear ablation (Asako et al., 2005). Reductions of cell size have also been observed in brainstem auditory nuclei of humans following adult-onset deafness (Moore et al., 1997). In animals, the reduced cell volumes following the various manipulations of peripheral input have been observed mainly in the AVCN, although one recent study reported a decrease in the soma area of DCN tuberculoventral cells 2 weeks following cochlear ablation (Asako et al., 2005). The cell volumes of fusiform cells in the DCN appear unaffected by these manipulations (Asako et al., 2005), probably reflecting the relatively stronger share of supportive input from descending and intrinsic pathways (Kane and Finn, 1977; Kane and Conlee, 1979). Reductions in cell size could lead to changes in the shape of action potentials, possibly decreasing their durations. There is evidence that neurons with shorter duration spikes have weaker synaptic strength, and thus weaker neurotransmitter release

than larger neurons (Sabatini and Regehr, 1997); postsynaptic neurons might therefore be expected to have decreased firing rates. Thus, even if most DCN cells do not themselves change in volume, their activity could be affected by reductions in cell volumes of AVCN neurons, since some neurons from the AVCN project directly to DCN neurons. Ventrotubercular neurons are located in the AVCN and posteroventral cochlear nucleus (PVCN) and project to the DCN where they influence the levels of neuronal activity. For example, Evans and Nelson (1973) observed inhibition of DCN neurons in response to electrical stimulation of the AVCN. Zhang and Oertel (1993) distinguished two types of stellate cells in the VCN, having opposite effects on DCN neurons. They found that D-stellate cells have an inhitory effect on DCN tuberculoventral cells whereas T-stellate cells have an excitatory effect. Tuberculoventral cells, also known as vertical or corn cells, inhibit DCN principal neurons (Voigt and Young, 1980; Young and Voigt, 1982; Zhang and Oertel, 1993). If the cells that are excitatory to DCN neurons shrink after loss of primary afferent input, the effect might be a decrease in spike duration and weaker synaptic strength producing a decrease in their excitatory effects on DCN neurons. Shrinkage may also decrease the metabolic capacity of neurons to process neurotransmitters for release. If shrinkage involves VCN cells that normally have an inhibitory influence on DCN neurons, the effect might be a reduction in the strength of inhibition of DCN neurons. This could lead to a disinhibition of some DCN cells with a resulting increase in their spontaneous activity.

The role of neural plasticity as a basis of hyperactivity The importance of neural plasticity in the etiology of tinnitus has been discussed in several recent reviews (Syka, 2002; Eggermont and Roberts, 2004; Kaltenbach et al., 2005; Møller, 2006) (see Chapter 3). Plastic alterations have been identified in the DCN following exposure to several types of tinnitus-inducing agents, including intense sound, aminoglycosides, cisplatin and sodium salicylate.

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Most of the changes reported have been observed at the synaptic level, although this may be due partly to the fact that synapses have been the focus of these studies. Plastic changes also occur at other cellular levels, such as the intracellular environment and cell membranes. Below is a summary of the relevant research findings.

Changes in neurotransmitter release/uptake Tinnitus often develops after injury or destruction of the eighth nerve, and there is evidence that this type of trauma can trigger changes in the balance of inhibitory and excitatory neurotransmission in all subdivisions of the CN. In the DCN, the release of the inhibitory transmitter, glycine, was reduced by 40% and its uptake was increased by 50%, 59 days after cochlear ablation (Suneja et al., 1998a; Potashner et al., 2000). The decrease continued until at least 145 days of survival when the glycine release dropped to 50% of the pre-ablation level. In the DCN decreases in the number of glycine-immunoreactive puncta, presumably representing presynaptic terminals, were observed on the somata of fusiform cells 14 days following bilateral cochlear ablation (Asako et al., 2005); decreases were also observed on the somata of spherical, globular bushy, stellate multipolar and radiate cells of the VCN. Some of these changes would presumably weaken inhibitory effects on fusiform cells, thus potentially raising their levels of spontaneous activity. On the other hand, increases in spontaneous activity could also result from increased release of excitatory transmitters. Transmitters that are excitatory to DCN neurons include glutamate or aspartate, acetylcholine and possibly serotonin. Large declines in the release of the excitatory transmitters, glutamate and D-aspartate occur in the CN after cochlear ablation (Potashner et al., 1997; Wenthold and Gulley, 1977). In the DCN the release of D-aspartate, showed a 50% decline from 2 to 145 days following ablation of the ipsilateral cochlea (Potashner et al., 1997). The decreases in D-aspartate release were even larger in the VCN, although these tended to recover to control levels by 59 days post-ablation (Potashner

et al., 1997). Thus, no evidence of a net increase in glutamate release was observed in any subdivision of the CN following cochlear ablation. However, increases in the release of aspartate have been observed in chinchillas exposed to intense noise (Muly et al., 2004). The ventral part of the DCN showed a nearly three-fold increase in evoked aspartate release, 7 days after noise exposure; this was followed by a nearly complete decline at 14 days post-exposure, then by a large increase in release to more than twofold above the control level 90 days after exposure. The dorsal part of the DCN showed only decreases in aspartate release following noise exposure. Noise exposure thus seems to have different effects on neurotransmission from those caused by ablation. This is consistent with the recent finding that DCN hyperactivity caused by intense sound exposure is different from that caused by outer hair cell loss, even when the volume and spread of hair cell loss is about the same (Carron et al., 2006). Although no direct measures of acetylcholine release following deafferentation have been reported, there is evidence for an increase in choline actetyl transferase (ChAT), the enzyme of acetylcholine synthesis, after intense sound exposure (Jin et al., 2006). Such increases were observed mostly in the granule cell region and are suggestive of an increase in cholinergic transmission to granule cells. Plastic alterations have also been observed in serotonin activity in the DCN following noise exposure (Cransac et al., 1998). This transmitter is released by non-auditory inputs to the CN from the dorsal raphe nucleus (Klepper and Herbert, 1991; Thompson et al., 1995; Thompson and Thompson, 2001). Both excitatory and inhibitory effects of serotonin on CN neurons have been reported, although mainly neurons in the VCN have been studied and these effects were found to be mostly inhibitory. There is a relatively greater share of serotonergic input to the DCN, although its influence on DCN neurons has yet to be established. Upregulation of serotonin activity in the DCN were found to increase with the level of noise exposure (Cransac et al., 1998). This upregulation might contribute to the emergence of hyperactivity.

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Changes at the receptor level There are numerous indications that receptors for excitatory and inhibitory transmitters may be altered by manipulations similar to those that cause changes in neurotransmitter release. Suneja et al. (1998b) found evidence for changes in glycine receptor expression in all three subdivisions of the CN after unilateral cochlear ablation. In the deep layer of the DCN, binding of 3H-strychnine, an antagonist of the glycine receptor, was unchanged at 2–7 days post-ablation, slightly increased at 31 days and decreased by 25% at 60 days postablation. In the fusiform cell layer, 3H-strychnine binding displayed a downward trend up to 2 months after ablation. Changes in binding to AMPA (alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid) receptors for glutamate or aspartate following cochlear ablation have also been studied in auditory brainstem nuclei, including each of the three layers of the DCN (Suneja et al., 2000). The molecular and fusiform cell layers of the ipsilateral DCN showed only marginal increases in AMPA binding 2 days after ablation. However, increases in AMPA binding were significant in the deep layer of the DCN 7 days after ablation, but at 60 days, binding in the deep layer was slightly decreased. Cochlear ablation can also trigger rearrangement in the distribution of AMPA receptors on DCN fusiform cells (Rubio, 2006). These changes are indicative of alterations in the number and function of glutamate receptors and could contribute to the changes in the level of spontaneous activity of postsynaptic neurons following cochlear insult. Various lines of evidence point to changes in receptors for the excitatory transmitter, acetylcholine, following loss of normal cochlear input. There are both nicotinic and muscarinic receptors for acetylcholine in the CN (Chen et al., 1994; Morley and Happe, 2000), although much larger changes in DCN spontaneous activity are induced by muscarinic than by nicotinic agents (Chen et al., 1994). Previous noise-exposure causes enhancements in the responses of DCN neurons to the cholinergic agonist, carbachol. Such enhancements have been observed in the superficial DCN (molecular and fusiform cell layers)

both in vitro (Chang et al., 2002) and in vivo (Kaltenbach and Zhang, 2006). Neurons with the properties of cartwheel cells (bursting activity) showed enhanced excitatory responses to carbachol 7–39 days following noise exposure, whereas multiunit ensembles recorded from the fusiform cell layer showed enhanced inhibitory responses. The enhanced excitation of putative cartwheel cells may result from an upregulation of acetylcholine receptors in the granule cell regions. Granule cells provide the main excitatory input to cartwheel cells, and recent evidence indicates that loss of cochlear input to the CN can cause an increase in the number of acetylcholine receptors in the granule cell regions of the CN and in the fusiform cell layer (Jin and Godfrey, 2006), which also includes granule cells. Although the identities of all the cell types that become hyperactive are not yet known, the available evidence suggests that both cartwheel cells and fusiform cells are likely sources (Brozoski et al., 2002; Chang et al., 2002; Kaltenbach and Zhang, 2006). Which cell type contributes more to the condition of hyperactivity may be dependent on the state of activation of granule cells, which likely depends on the number of acetylcholine receptors and the volume of cholinergic inputs to the granule cell domain.

Changes in the number of synapses Changes in transmitter release and uptake as well as in the number of receptors, as just described, may ultimately reflect changes in the number of active synapses. Several recent papers report that loss of cochlear input, due either to cochlear ablation or noise-induced hearing loss, leads to an increase in the number of cholinergic synapses in the CN (Meidinger et al., 2006). Thus staining for GAP-43, a marker for synaptic remodeling, was found to increase in much of the VCN following cochlear ablation (Illing et al., 1997, 2005) and noise exposure (Michler and Illing, 2002; Illing et al., 2005; Meidinger et al., 2006). No mention was made of such changes in the DCN or in the granule cell regions. However, Jin et al. (2006) reported increases in the expression of ChAT following intense tone exposure in the granule cell

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regions of the hamster DCN. This enzyme is present in the presynaptic terminals of cholinergic neurons where it is involved in the synthesis of acetylcholine. Jin et al. (2006) interpreted the increase in ChAT expression as indicative of a possible increase in the number of cholinergic terminals in the granule cell regions. Sprouting of new synapses in the DCN and VCN following cochlear ablation has been reported, although the available evidence indicates that the degree in the adult DCN is probably much less than in the VCN (Benson et al., 1997). In the DCN of guinea pigs an increase in the number of synapses was observed in the deep layer 4 days after unilateral cochlear ablation, despite loss of primary afferents at this time; no changes in the number of synapses was apparent in the DCN from 7 days through 161 days postablation. In contrast, evidence for synaptogenesis in the AVCN of the same animals was more apparent after 7 days postablation, and followed a period of degeneration of primary afferents at 4 and 7 days (Benson et al., 1997). Evidence for synaptic sprouting in the VCN of chinchillas after acoustic overstimulation has been reported in several studies (Bilak et al., 1997; Muly et al., 2002; Kim et al., 2004a, b). Kim et al. (2004a, b) examined patterns of synaptic change in the AVCN and PVCN over a period of 6–8 months following a single 3-h exposure to octave band noise (centered at 4 kHz) at a level of 108 dB SPL. In the AVCN, degeneration of excitatory and inhibitory synaptic terminals was observed around the somata and dendrites of globular bushy cells as well as in the neuropil. This degeneration was followed and accompanied by the emergence of new synapses distinguished by their small size and lower number of vesicles. The new synapses appeared to fill in the vacated spaces left by loss of the original terminals. Both degeneration and synaptogenesis were observed chronically and found to continue for the entire 8 months of observation. Degeneration of excitatory and inhibitory synapses was also seen in the PVCN. The recovery of synapses around the somata of PVCN neurons and in the neuropil was less complete for the inhibitory than for the excitatory terminals. This led to a net decrease in the inhibitory input to

these elements. This would seem to favor an increase in neuronal excitation. Whether a similar net loss of inhibitory synapses occurs in the DCN has not yet been established, although the loss of glycine receptors in the DCN following cochlear ablation, as described above, would be consistent with a decrease in inhibitory synapses. DCN hyperactivity could result from a loss of glycine receptors and/or glycinergic synapses from the DCN, from a loss of inhibitory synapses of VCN neurons that project to DCN neurons or from an increase in excitatory input from the VCN. It is also possible that DCN hyperactivity may be associated with hyperactivity in the VCN. However, no studies have yet reported increases in spontaneous activity in the VCN after intense noise exposure or cochlear insult. Future studies are needed to shed more light on these possibilities.

Changes in ion conductance Changes in ion conductances are not necessarily independent of the changes discussed above. Indeed, changes in ion channels are to be expected if there are changes in synaptic receptors since many receptors are ionotropic and therefore include ion channels as part of their tertiary structures. Other receptors indirectly control ion channels that are distributed elsewhere, either by their influence on intracellular signaling pathways or by their effects on membrane voltages. Changes in ion conductance are of special interest and are considered here because of their role in controlling ion permeability of the cell membrane. This function determines the resting membrane potential, the threshold of spike generation, the duration of action potentials and the time course of recovery from action potentials. They can thus be considered pivotal in determining the excitability of neurons and, therefore, their spontaneous discharge rates. Despite their importance, relatively little is known about the effects of tinnitus-inducing manipulations on ion channels of DCN neurons. Several published studies have focused on the VCN, which is relevant to activity in the DCN, as it receives direct input from the VCN.

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Francis and Manis (2000) examined the effects of bilateral cochlear ablation on the membrane properties of neurons in the AVCN. The cochleae were removed in 1-month-old rats, and the animals were studied electrophysiologically in brain slices 2 weeks later. Deafferented AVCN neurons showed more depolarization and smaller action potentials, smaller afterhyperpolarizations and shorter membrane time constants compared to those in control animals. Other neurons showed evidence for increases in input resistance. The authors’ explanation for these changes was that they might reflect alterations in cell size or changes in the composition, properties or spatial distribution of voltage-dependent ion channels. However, only a few published studies have examined changes in ion channels in the VCN after cochlear ablation. Caminos et al. (2005) found no changes in the expressions or spatial distributions of Kv1.1 or Kv1.2 potassium channels in AVCN neurons 10 days after bilateral cochlear ablation in young adult rats. Leao et al. (2005) found no difference in Ih channels or membrane excitability when spherical bushy cells in the AVCN of normal hearing CBA mice were compared with those of congenitally deaf mice. A more recent study reported changes in the expression of the two-pore domain potassium channel in the rat cochlear nucleus following bilateral cochlear ablation (Holt et al., 2006). It is not yet clear in which subdivision these changes occur, but the results revealed a sustained decrease in transcripts over three recovery times for three subunits (TASK-1, 2 and 5). Transcripts for three other subunits (TWIK-2, TREK-1 and TREK-2) were expressed at normal levels 3 days after ablation but decreased at 3 weeks and 3 months. Two subunits (TRAAK and TASK-3) were decreased at 3 days, but returned to their normal levels of expression at the 3-week and 3-month time frames. The lattermost changes thus suggest plastic changes of the two-pore domain potassium channel in response to deafferentation. Such changes could be important factors controlling both spontaneous activity and responses to sound, since one function of the two-pore domain potassium channel is to set the resting membrane potential.

Changes in cell signaling proteins Ultimately, disturbances in transmitter release, receptors binding, numbers of synapses and numbers of ion channels may reflect alterations in regulatory pathways and intracellular signaling cascades. Changes in neurotransmission caused by cochlear ablation are accompanied by alterations in the expression of numerous enzymes of signal transduction pathways, such as cyclic-AMP dependent protein kinase (PKA), protein kinase C (PKC), calcium/calmodulin-dependent protein kinase (CaMKII), (Garcia et al., 2000; Zhang et al., 2003, 2004; Yan et al., 2006), phosphorylated cAMP reponse element-binding protein (CREB) (Illing and Michler, 2001; Michler and Illing, 2003; Mo et al., 2006), extracellular signalregulated kinase (ERK) and stress-activated protein kinase (SAPK) (Suneja and Potashner, 2003). Some of these play roles in regulating transmitter release or receptor binding (Yan et al., 2006). Changes in the numbers of synapses following deafferentation are also accompanied by increases in growth factors in the cochlear nucleus. Examples include increases in neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) after cochlear ablation (Suneja et al., 2005), and increases in fibroblast growth factors after acoustic injury (Smith et al., 2002). These changes are triggered by the loss of synapses and provide stimulation for the growth of new synapses.

Net effects of altered input to the cochlear nucleus (Fig. 3) Decreases in inhibition To summarize, several lines of evidence indicate that manipulations that result in loss of normal input (both anatomical and functional) to the auditory brainstem cause a decrease in inhibition in the DCN that is greater than the decreases in excitation (Fig. 3). Such decreases are observed in the DCN as declines in glycine release (Suneja et al., 1998a; Potashner et al., 2000) and reductions of glycine immunoreactivity after cochlear ablation (Asako et al., 2005), and greater reductions in

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Fig. 3. Summary of changes likely to contribute to the generation of hyperactivity in the DCN. The changes shown here have been shown to occur in the DCN as a consequence of noise exposure, cochlear ablation and/or other manipulations that are tinnitus inducing.

the number of inhibitory synapses than excitatory synapses after either cochlear ablation or noise exposure (Kim et al., 1997; Morest et al., 1998; Muly et al., 2002). Such changes likely involve plastic readjustments to loss of input since they continue to occur long after the period when loss of primary afferent fibers is observed. Increased spontaneous activity in the DCN might be a direct consequence of this loss of glycinergic function. This seems likely given that blockade of glycine receptors in animals with intact primary afferent input to the DCN can produce increases in spontaneous activity of DCN principal neurons and cartwheel cells (Caspary et al., 1987; Evans and Zhao, 1993; Zhang and Oertel, 1994; Davis and Young, 2000). These considerations suggest that loss of inhibitory influence on DCN neurons, particularly on the fusiform cells, may be the dominant mechanism underlying the emergence of hyperactivity in the DCN after peripheral insult.

Increased excitation As just explained, increases in excitation might be expected based on the observations that loss of inhibition is greater than the loss of excitation. However, further increases in excitation seem likely because loss of inhibition is associated with increases in the number of excitatory synapses. Evidence for increased excitation in the DCN, after loss of normal anatomical or functional input

from the cochlea, includes increases in AMPA binding after cochlear ablation and increases in glutamatergic transmitter release following noise exposure (Muly et al., 2004). The increases in transmitter release were apparent in the ventral part of the DCN 7 and 90 days after the initial insult, but were reduced at 14 days. These changes in glutamatergic transmission reflect synaptic plasticity and cannot explain the persistence of hyperactivity at 14 days (Kaltenbach et al., 2000). However, excess glutamate release could cause further injury to DCN neurons through the mechanism of excitotoxicity (Fig. 2B), which could, in turn, result in damage to inhibitory interneurons, such as cartwheel and stellate cells. Permanent loss or damage to these cells could cause chronic disinhibition of fusiform cells, leading to chronic increases of fusiform cell activity. The increase in glutamatergic neurotransmission thus seems likely to be an important mechanism contributing to the generation of tinnitus-related hyperactivity in the DCN. Chronic hyperactivity also has a cholinergic component, which probably acts on the DCN through granule cells. There is evidence for increased binding to cholinergic receptors after cochlear ablation (Jin and Godfrey, 2006) and increased sensitivity of the DCN to cholinergic agonists after noise exposure (Chang et al. 2002; Kaltenbach and Zhang, 2006). These increases would tend to raise the firing rates of cartwheel and stellate cells, the targets of granule cells, thus enhancing their contribution to hyperactivity.

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Increased membrane excitability Evidence has been presented for changes in the expression of ion conductance channels that influence cellular excitability levels independent of neurotransmitter receptors. Chief among these is the two-pore domain potassium channel. Expression of this channel has been shown to decrease in the CN following cochlear ablation. It is not yet clear in which subdivision this decrease occurs or whether this change also occurs following other tinnitus-inducing manipulations. However, changes in the two-pore domain potassium channel may be an important factor contributing to hyperactivity, in view of its role in controlling a cell’s excitability and in altering the resting membrane potential (Millar et al., 2000). Summary and conclusions The evidence presented above supports a role of the DCN in the generation and modulation of tinnitus. There is growing support for the hypothesis that at least some forms of tinnitus are linked to increased spontaneous activity in the DCN. Animal studies have shown that loss or reduction of normal peripheral input, such as occurs following intense noise exposure, cochlear ablation and ototoxic injury, causes both decreases in inhibitory (glycinergic) and increases in excitatory (glutamatergic and cholinergic) neurotransmission in the DCN. These changes arise as a consequence of degeneration of synapses, expression of synaptic plasticity and probably also because of injuries caused by excitotoxicity. Changes in the ion conductance properties of neurons, outside synapses, may also contribute to the observed changes in spontaneous activity, although this possibility is only now beginning to be explored. It seems reasonable to expect that knowledge about changes in cellular processes that may be involved in causing the abnormal neural activity that results in tinnitus will stimulate new approaches for the treatment of tinnitus. Acknowledgments This work was supported by funds from NIDCD R01 DC003258. I would also like to thank my

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