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Spontaneous hyperactivity in the auditory midbrain: Relationship to afferent input
Hearing Research 295 (2013) 124e129
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Hearing Research journal homepage: www.elsevier.com/locate/heares
Spontaneous hyperactivity in the auditory midbrain: Relationship to afferent input Donald Robertson*, Christofer Bester, Darryl Vogler, Wilhelmina H.A.M. Mulders The Auditory Laboratory, School of Anatomy, Physiology and Human Biology, M311, The University of Western Australia, 35 Stirling hwy, Crawley, Western Australia 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2011 Received in revised form 31 January 2012 Accepted 2 February 2012 Available online 13 February 2012
Hyperactivity in the form of increased spontaneous firing rates of single neurons develops in the guinea pig inferior colliculus (IC) after unilateral loud sound exposures that result in behavioural signs of tinnitus. The hyperactivity is found in those parts of the topographic frequency map in the IC where neurons possess characteristic frequencies (CFs) closely related to the region in the cochlea where lasting sensitivity changes occur as a result of the loud sound exposure. The observed hyperactivity could be endogenous to the IC, or it could be driven by hyperactivity at lower stages of the auditory pathway. In addition to the dorsal cochlear nucleus (DCN) hyperactivity reported by others, specific cell types in the ventral cochlear nucleus (VCN) also show hyperactivity in this animal model suggesting that increased drive from several regions of the lower brainstem could contribute to the observed hyperactivity in the midbrain. In addition, spontaneous afferent drive from the cochlea itself is necessary for the maintenance of hyperactivity up to about 8 weeks post cochlear trauma. After 8 weeks however, IC hyperactivity becomes less dependent on cochlear input, suggesting that central neurons transition from a state of hyperexcitability to a state in which they generate their own endogenous firing. The results suggest that there might be a “therapeutic window” for early-onset tinnitus, using treatments that reduce cochlear afferent firing. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Elevated levels of spontaneous neural firing (spontaneous hyperactivity) have been described in a number of brain regions after treatments that are known to induce tinnitus in humans and that cause behavioural changes consistent with tinnitus in animal experiments. Numerous animal models have shown a consistent pattern of hyperactivity in the inferior colliculus (IC) after unilateral partial deafness induced by acoustic trauma and other forms of damage (Bauer et al., 2008; Dong et al., 2009,b; Mulders and Robertson, 2009). This hyperactivity appears to be associated with altered patterns of gene expression in the IC, including reduced expression of elements of inhibitory, notably GABAergic synaptic machinery and of Kþ ion channels that stabilize membrane potentials (Dong et al., 2009, 2010a,b). Although Abbreviations: IC, inferior colliculus; VCN, ventral cochlear nucleus; DCN, dorsal cochlear nucleus; PL, primary-like; PLn, primary-like with notch; CF, characteristic frequency; CAP, compound action potential; GPIAS, gap-induced suppression of acoustic startle response; MOCS, medial olivocochlear system; PSTH, peristimulustime histogram; CNS, central nervous system; GABA, g amino butyric acid. * Corresponding author. Fax: þ61 8 6488 1025. E-mail addresses: [email protected], [email protected] (D. Robertson). 0378-5955/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2012.02.002
a causal relationship between IC hyperactivity and behavioural tinnitus has not been definitively established, the central role of this structure in the ascending auditory pathways means that midbrain hyperactivity continues to be used as a promising model for the neural substrates of this potentially debilitating disorder. An important question is to what extent the hyperactivity in the IC (and possibly therefore, that seen in even higher parts of the auditory pathway such as the auditory cortex) is a result of altered intrinsic properties of the IC neurons and their local circuitry, and how much is dependent on input from lower stages of the pathway, such as the cochlear nucleus and even the cochlea itself. There are numerous reports that cochlear trauma of the type that leads both to behavioural tinnitus and to midbrain hyperactivity, also produces hyperactivity in the dorsal subdivision of the cochlear nucleus (DCN) (Kaltenbach et al., 2000, 2004) but until recently, the ventral subdivision (VCN) had not been directly investigated. With regard to the role of the cochlea, little attention has been paid to the relationship between spontaneous cochlear neural activity, tinnitus and IC hyperactivity, aside from the fact that some loss of cochlear afferent drive to the CNS appears to be a common precipitating cause of tinnitus. Indeed there is a generally pervasive view that while cochlear trauma may initiate the chain of events that eventually leads to tinnitus and the associated abnormal central neural
D. Robertson et al. / Hearing Research 295 (2013) 124e129
activity, tinnitus itself is essentially a central phenomenon, independent of ongoing background afferent input from the cochlea for its maintenance. In this paper, we review some of our recent published findings on hyperactivity in the brainstem and midbrain and also present some new data. We seek to address several questions: 1) how precise is the relationship between changes in cochlear afferent neural sensitivity and the features of hyperactivity in the IC? 2) is the VCN also a potential source of abnormal input to the IC? and 3) how dependent is central neural hyperactivity on the presence of ongoing spontaneous firing of cochlear primary afferent neurons? 2. Methods For all the experiments reported here, detailed electrophysiological methods have already been published (Mulders and Robertson, 2009, 2011; Vogler et al., 2011). In brief, groups of anaesthetized pigmented guinea pigs were subjected to pure tone acoustic trauma in one ear. After recovery times ranging from 1 to 12 weeks, terminal experiments were performed, again under general anaesthesia. In the terminal experiments, microelectrode recordings were made from large numbers of single neurons (70e110 per animal) in the central nucleus of the IC contralateral to the traumatized cochlea, or from single neurons in the ipsilateral VCN. In the case of IC recordings, the depth at which each neuron was encountered was recorded, as well as characteristic frequency (CF) and spontaneous firing rate estimated from a 10 s sample in the absence of external acoustic stimulation. For cochlear nucleus recordings, CF and spontaneous firing rate were recorded for each neuron encountered, as well as peristimulus-time histograms (PSTHs) in order to enable classification of neuron response type. In both IC and VCN recordings, all the data discussed were obtained from true single neuron recordings and not, as is often the case in several other studies, from multiunit or “ensemble” activity. In some terminal experiments, the spontaneous neural activity emanating from the cochlea was acutely silenced using either complete cochlear ablation, or cochlear perfusion of drugs that blocked either neurotransmitter release from inner hair cells or the postsynaptic receptors responding to the inner hair cell neurotransmitter (Mulders and Robertson, 2009). It is important to stress that these methods of cochlear silencing were acute, not chronic, and were performed on animals after the recovery period from initial acoustic trauma had taken place. Hence in these experiments, we observed the immediate effects of such treatments on neural hyperactivity that had already developed in the IC. We have also studied the immediate effects of electrical stimulation of the medial olivocochlear system (MOCS) on IC hyperactivity (Mulders et al., 2010). This was achieved by delivering trains of shocks to the MOCS axons at the floor of the IVth ventricle, while continuously recording the spontaneous firing of individual hyperactive neurons in the IC. The contribution of the intracochlear action of the MOCS system to the effects seen was evaluated by simultaneous intracochlear perfusion of strychnine, a potent reversible blocker of MOCS action on outer hair cells. Behavioural evaluation of tinnitus was performed using the gap suppression of acoustic startle method (GPIAS) reported in detail by Yang et al. (2007). An important consideration with this technique is that only unilateral acoustic insults can be routinely employed to induce hyperactivity and tinnitus, since the test relies on the subject responding to a gap in relatively low level background noise. Reduced sensitivity binaurally could make the noise and gap less audible and so artifactually bias the results. In all animals therefore, normal thresholds in the untreated cochlea were evaluated by recording of the compound action potential (CAP) audiogram prior to the final terminal experiments.
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3. Results 3.1. Features of IC hyperactivity Our animal model consistently shows the presence of neuronal hyperactivity (increased mean spontaneous firing rates) in IC. This hyperactivity is not an immediate reaction to the cochlear trauma, and takes time to develop. It is not evident immediately after cochlear trauma (Mulders and Robertson, 2011) but is significant from 1 week afterwards. The level of hyperactivity increases further between 1 and 2 weeks post cochlear trauma and is then maintained for up to 12 weeks post trauma (the longest recovery period that we have used in these experiments). In this animal model, mean spontaneous firing rates are close to zero in sham operated animals and in animals in which it is measured immediately after cochlear trauma. Mean spontaneous firing rates then increase to average values of 8e12 spikes/s at 1e12 weeks post exposure. As has been reported in other brain regions, hyperactivity is not uniform across the neuronal population in any given CF region, with many neurons in IC still showing spontaneous firing rates that are not higher than normal. Instead, hyperactivity is manifested as greatly increased firing rates in a relatively small subset of neurons. One of the questions still to be answered in IC is in what manner the subset of hyperactive neurons is different from the majority of IC neurons that do not show hyperactivity. In this animal model, it can be shown that hyperactivity in the IC can be accompanied by behavioural evidence of tinnitus. Fig. 1 shows the results obtained using the GPIAS test in a separate group of animals (n ¼ 4) between 7 and 12 days after inducing a high frequency hearing loss. The results after trauma show a reduction in gap suppression of startle (in this instance the gap did not significantly reduce the startle response) when using bandpass background noise centred at 14 kHz, but not when the noise is centred at 8 kHz. These results strongly suggest that these animals experience tinnitus with a spectral quality above about 10 kHz. This result might be predicted from the previous electrophysiological data which consistently showed after trauma localized to the high frequency region of the cochlea, that hyperactivity in the IC is significant for neurons with CF’s above 10 kHz, but not below (Mulders and Robertson, 2009, 2011; Mulders et al., 2011). The result is consistent with, but does not prove, a causal relationship between hyperactivity and tinnitus. The relationship between the detailed features of the peripheral cochlear trauma and the eventual distribution of hyperactivity in the IC was investigated in more detail by using two different
Fig. 1. Results of behavioural demonstration of tinnitus in group of guinea pigs (n ¼ 4) tested before and 7e12 days after trauma to the high frequency region of the cochlea. Vertical axis, % gap-induced suppression of startle using bandpass noise centred at 8 kHz (open bars) and 10 kHz (solid bars). Error bars (S.E.M). Note greatly reduced gap suppression of startle for 10 kHz background noise and lack of change for 8 kHz background noise.
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traumatizing exposures (5 kHz and 10 kHz at 124 dB SPL for 2 h) in separate groups of animals. The peripheral damage was estimated by careful measurement of the cochlear compound action potential (CAP) threshold audiogram. Fig. 2A and B shows that these two exposures result in clearly different patterns of peripheral sensitivity change. Fig. 2C and D shows the detailed relationship between these CAP threshold changes and the CF distribution of spontaneous firing rates in the IC. The latter is displayed as a running average of the spontaneous firing rates of all individual neurons encountered as a function of their CF. Fig. 2E shows the distribution of the hyperactivity per frequency region. It is clear from these data, that there is a rather tight relationship between the “existence region” of hyperactivity in the tonotopic map of the IC, and the frequencies at which peripheral CAP thresholds have been affected by the prior noise exposure. 3.2. Hyperactivity in VCN Similar forms of cochlear trauma to that employed in our studies of IC hyperactivity have been shown by others to induce hyperactivity in the dorsal subdivision of cochlear nucleus (DCN) (Kaltenbach et al., 2000, 2004). Until recently, the ventral subdivision (VCN) had not been directly investigated in this manner, even though the VCN provides a major source of indirect input to the IC. Therefore we have used our animal model to record from large numbers of single neurons in the VCN (Vogler et al., 2011). Clear evidence of hyperactivity was obtained, with average spontaneous firing rates being increased by 104% relative to sham operated animals. Fig. 3 shows the distribution of spontaneous rates across the range of CFs encountered in VCN. Interestingly, hyperactivity in VCN was more widespread in the tonotopic domain than was seen in IC, with significant elevation of mean spontaneous firing rates being found both when neurons were grouped according to CFs below and above 10 kHz. In this study, in addition to CF and spontaneous firing rates, neuron response type was also determined and it was found that only onset and primary-like neurons showed spontaneous firing rates that were significantly greater than in sham operated animals of the same age. When primary-like neurons were further categorized into primary-like (PL) or primary-like with notch (PLn) (Winter and Palmer, 1990), the greatest increase in spontaneous firing rates was seen for the PLn category, but this increase was not significant, probably because of the relatively small sample sizes when neurons are placed in these sub-categories of response type. Similarly, although a significant increase in spontaneous rate was found when all types of onset neurons were pooled, the sample size was not sufficient to indicate whether this rise was confined to particular onset response sub-categories. 3.3. Dependence on cochlear afferent drive We have shown that hyperactivity in the IC in our guinea pig model can be abolished, or greatly reduced, by silencing the spontaneous firing of primary cochlear afferent neurons (Mulders and Robertson, 2009). This has been demonstrated in two ways. In an initial set of experiments, after sampling hyperactivity from a large number of IC neurons, the originally traumatized cochlea contralateral to the IC was acutely ablated, and in the same animals, a further sampling was undertaken. It was clearly demonstrated using this method, that the hyperactivity was no longer present after such acute cochlear ablation. The acute ablation technique could have suffered from a number of confounding factors, and hence, in a further set of experiments, we silenced the contralateral cochlea using local cochlear cooling and by using intracochlear
perfusion of blockers of afferent firing (kainic acid and cobalt) while continuously recording the spontaneous firing of single hyperactive IC neurons. These experiments confirmed the results of the cochlear ablation studies, showing that the hyperactivity in IC neurons required intact neural activity from the cochlea to be maintained. These experiments on cochlear silencing were initially performed after 2 weeks recovery. We have also investigated the dependence of IC hyperactivity on cochlear integrity at recovery times up to 12 weeks post acoustic trauma (Mulders and Robertson, 2011). In these studies, it was found that the hyperactivity could be abolished by acute cochlear ablation up to 6 weeks post acoustic trauma. However, from 8 to 12 weeks post acoustic trauma, although cochlear ablation did cause some reduction in hyperactivity in the IC, this was not statistically significant. Hence the dependence of IC hyperactivity on continuing input from the contralateral cochlea appears to decrease the longer the hyperactivity has been present. 3.4. Effect of MOCS stimulation We have also shown that electrical stimulation of the MOCS transiently suppresses hyperactivity in single hyperactive IC neurons (Mulders et al., 2010). Intracochlear perfusion of strychnine showed that in most hyperactive IC neurons, this MOCSmediated suppression was almost entirely attributable to the intracochlear action of the MOCS. In view of the known action of MOCS in suppressing spontaneous cochlear neural output, this result provides additional support for the previous findings that at least at early stages in its development, central hyperactivity is dependent on continued neural drive from the cochlea. 4. Discussion In the guinea pig model reported here, central hyperactivity is not present within the first few hours post cochlear trauma (the time taken to collect sufficient single neuron data immediately after a cochlear acoustic trauma). Clearly therefore the model cannot provide a neural basis for tinnitus of sudden onset, since it appears to involve a progressive change in central neural activity that is observed, after a recovery period of 1 week post cochlear trauma. This limitation aside however, the results on IC hyperactivity show several interesting features that may assist in understanding the development of some forms of tinnitus, and their possible treatment. First the single neuron data both in the IC and the VCN reveal that hyperactivity is found only in a subset of neurons in any particular location. It is worth noting that such a conclusion could not be readily obtained from multiunit and ensemble recordings. A similar basic finding has been found in other brain regions as well, notably in the DCN in which it has been reported that only fusiform cells receiving convergent somatosensory and auditory input exhibit hyperactivity after cochlear trauma (Shore, 2011; Shore et al., 2008). These results in general suggest that the tinnitus percept may be the result of abnormal activity in discrete neural channels. Our finding of hyperactivity in primary-like and onset neurons in VCN is also consistent with this notion and it is interesting to note that recent studies suggest that abnormal excitability of the spherical bushy cell pathway from VCN (neurons which show primary-like characteristics) may be a key neural feature associated with tinnitus (Melcher this volume). These results could have implications for possible treatments, since attempts to reduce overall neural excitability, or increase overall inhibitory activity, may lack the specificity required to target the particular neural populations involved.
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Fig. 2. A and B, Effects of two different noise exposure regimes (10 kHz (B) and 5 kHz (A) traumatizing tone, 124 dB SPL, 2 h duration) on cochlear neural thresholds 2 weeks post exposure. Data for 10 kHz exposure group published previously (Mulders et al., 2011). Vertical axis, change in cochlear compound action potential threshold (CAP threshold) compared to pre-exposure thresholds. Asterisks denote changes that are statistically significant compared to pre-exposure thresholds (n ¼ 4 animals in each group). Data for 5 kHz group derived from 437 neurons. C and D, Relationship between CAP threshold changes (solid circles and line with error bars) and single neuron spontaneous firing rates in IC (continuous solid line). Single neuron firing rates displayed as a running average (n ¼ 29) of pooled data from all animals in each group. Data for 10 kHz exposure group published previously (Mulders et al., 2011). E, Histogram of mean spontaneous firing rates in IC per CF region in sham (white bars), 5 kHz (grey bars) and 10 kHz (black bars) acoustic trauma groups. *p < 0.05; **p < 0.01; #p < 0.001.
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Fig. 3. Mean spontaneous firing rates of neurons in VCN in sham (A) and acoustic trauma (B) groups. Data pooled from all neuron response categories and divided into 2 kHz CF bands. Solid circles and lines show average change in cochlear CAP thresholds after 2 weeks recovery, relative to initial thresholds in the two groups.
The finding that the pattern of hyperactivity in the IC matches remarkably well to the pattern of neural sensitivity changes in the cochlea (Fig. 2) is also of interest. Although we have not made detailed behavioural evaluations of tinnitus to see whether the perceived tinnitus spectrum shifts according to the shift in distribution of hyperactivity in these same animals, the result is consistent with observations in humans that the perceived tinnitus spectrum closely matches the pattern of threshold changes in the behavioural audiogram (Norena et al., 2002) and this strengthens the case for a possible causal relationship between hyperactivity and tinnitus. It should be noted however, that a close correspondence between peripheral threshold change, tinnitus and hyperactivity could be co-incidental. It has been reported that acoustic trauma can cause selective degeneration of higher threshold primary afferent neurons (Kujawa and Liberman, 2009), which would leave cochlear threshold unaltered. The most novel aspect of our results is the fact that cochlear silencing causes an immediate reduction of hyperactivity in the IC. This finding challenges the prevailing view that tinnitus, although it
is most commonly triggered by a cochlear event such as acoustic trauma, is fundamentally a maladaptive form of central plasticity in which the resulting abnormal neural activity is endogenously generated in central pathways. There is indeed ample evidence in humans and animal models that tinnitus can persist in the absence of cochlear afferent input to the brainstem (Kaltenbach, 2011; Zacharek et al., 2002). How can this be reconciled with our findings? Our working hypothesis (Fig. 4) does not rely on any suggestion that primary afferent spontaneous activity is elevated. Instead, we have suggested that the first stage of maladaptive plasticity is a hyperexcitability of central neurons that renders them more likely to generate action potentials in response to any remaining spontaneous afferent drive from the cochlea. In this early phase, elimination of the cochlear input will immediately eliminate the central hyperactivity. Our results show however, that there is a second, later phase, which we have dubbed “progressive centralization” (Mulders and Robertson, 2011) in which cochlear silencing is no longer completely effective. We suggest that this is the result of the central neurons becoming so excitable that they generate their own endogenous spontaneous activity. It is interesting to speculate on the mechanisms and the therapeutic implications of such “progressive centralization”. It may be that the initial cochlear trauma sets in motion a prolonged sequence of cellular events that is fixed in its progression. Alternatively, the early phase of hyperactivity may itself contribute to further maladaptive changes that lead to eventual centralization (Fig. 4). If the latter is the case, then temporarily reducing primary afferent firing early in the development of tinnitus might act to arrest, or even reverse, the progression to the more centralized state. The exploitation of this “therapeutic window” might form the basis of novel approaches to the treatment of tinnitus of recent onset. It is worth noting that the midbrain hyperactivity described in this review shows some significant differences from the behaviour of neurons of the cat auditory cortex after similar acoustic trauma. Spontaneous firing rates in auditory cortical neurons are not increased immediately after trauma (similar to our own results in IC), but hyperactivity does develop within 2 h post trauma and this hyperactivity appears to be no greater after more extended recovery periods (Norena and Eggermont, 2003, 2006). It is not clear therefore to what extent cortical hyperactivity may be independent of midbrain hyperactivity, and the implications of this for the notion of a “therapeutic window” targeting midbrain hyperactivity will require further investigation. Acknowledgements This work was supported by grants to D. Robertson and W. Mulders from Action on Hearing Loss, The Neurotrauma Research Program and the Medical Health and Research Infrastructure Fund. C. Bester and D. Vogler are currently recipients of Australian Postgraduate Awards. The authors are grateful to R. Salvi for advice and encouragement, D. Stolzberg for generous technical assistance and donation of the GPIAS software and I. Winter and I.M. Lloyd for providing single neuron software (Neurosound) and microelectrodes. References
Fig. 4. Schematic representation of proposed stages of development of IC hyperactivity.
Bauer, C.A., Turner, J.G., Caspary, D.M., Myers, K.S., Brozoski, T.J., 2008. Tinnitus and inferior colliculus activity in chinchillas related to three distinct patterns of cochlear trauma. J. Neurosci. Res. 86, 2564e2578. Dong, S., Mulders, W.H., Rodger, J., Robertson, D., 2009. Changes in neuronal activity and gene expression in guinea-pig auditory brainstem after unilateral partial hearing loss. Neuroscience 159, 1164e1174. Dong, S., Rodger, J., Mulders, W.H., Robertson, D., 2010a. Tonotopic changes in GABA receptor expression in guinea pig inferior colliculus after partial unilateral hearing loss. Brain Res. 1342, 24e32.
D. Robertson et al. / Hearing Research 295 (2013) 124e129 Dong, S., Mulders, W.H., Rodger, J., Woo, S., Robertson, D., 2010b. Acoustic trauma evokes hyperactivity and changes in gene expression in guinea-pig auditory brainstem. Eur. J. Neurosci. 31, 1616e1628. Kaltenbach, J.A., 2011. Tinnitus: models and mechanisms. Hear. Res. 276, 52e60. Kaltenbach, J.A., Zhang, J., Afman, C.E., 2000. Plasticity of spontaneous neural activity in the dorsal cochlear nucleus after intense sound exposure. Hear. Res. 147, 282e292. Kaltenbach, J.A., Zacharek, M.A., Zhang, J., Frederick, S., 2004. Activity in the dorsal cochlear nucleus of hamsters previously tested for tinnitus following intense tone exposure. Neurosci. Lett. 355, 121e125. Kujawa, S.G., Liberman, M.C., 2009. Adding insult to injury: cochlear nerve degeneration after “temporary” noise-induced hearing loss. J. Neurosci. 29, 14077e14085. Mulders, W.H., Robertson, D., 2009. Hyperactivity in the auditory midbrain after acoustic trauma: dependence on cochlear activity. Neuroscience 164, 733e746. Mulders, W.H., Robertson, D., 2011. Progressive centralization of midbrain hyperactivity after acoustic trauma. Neuroscience 192, 753e760. Mulders, W.H., Seluakumaran, K., Robertson, D., 2010. Efferent pathways modulate hyperactivity in inferior colliculus. J. Neurosci. 30, 9578e9587. Mulders, W.H., Ding, D., Salvi, R., Robertson, D., 2011. Relationship between auditory thresholds, central spontaneous activity, and hair cell loss after acoustic trauma. J. Comp. Neurol. 519, 2637e2647.
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Norena, A., Micheyl, C., Chery-Croze, S., Collet, L., 2002. Psychoacoustic characterization of the tinnitus spectrum: implications for the underlying mechanisms of tinnitus. Audiol. Neurootol. 7, 358e369. Norena, A.J., Eggermont, J.J., 2003. Changes in spontaneous neural activity immediately after an acoustic trauma: implications for neural correlates of tinnitus. Hear. Res. 183, 137e153. Norena, A.J., Eggermont, J.J., 2006. Enriched acoustic environment after noise trauma abolishes neural signs of tinnitus. Neuroreport 17, 559e563. Shore, S.E., 2011. Plasticity of somatosensory inputs to the cochlear nucleus e implications for tinnitus. Hear. Res. 281, 38e46. Shore, S.E., Koehler, S., Oldakowski, M., Hughes, L.F., Syed, S., 2008. Dorsal cochlear nucleus responses to somatosensory stimulation are enhanced after noiseinduced hearing loss. Eur. J. Neurosci. 27, 155e168. Vogler, D.P., Robertson, D., Mulders, W.H., 2011. Hyperactivity in the ventral cochlear nucleus after cochlear trauma. J. Neurosci. 31, 6639e6645. Winter, I.M., Palmer, A.R., 1990. Responses of single units in the anteroventral cochlear nucleus of the guinea pig. Hear. Res. 44, 161e178. Yang, G., Lobarinas, E., Zhang, L., Turner, J., Stolzberg, D., Salvi, R., Sun, W., 2007. Salicylate induced tinnitus: behavioral measures and neural activity in auditory cortex of awake rats. Hear. Res. 226, 244e253. Zacharek, M.A., Kaltenbach, J.A., Mathog, T.A., Zhang, J., 2002. Effects of cochlear ablation on noise induced hyperactivity in the hamster dorsal cochlear nucleus: implications for the origin of noise induced tinnitus. Hear. Res. 172, 137e143.
Contents lists available at SciVerse ScienceDirect
Hearing Research journal homepage: www.elsevier.com/locate/heares
Spontaneous hyperactivity in the auditory midbrain: Relationship to afferent input Donald Robertson*, Christofer Bester, Darryl Vogler, Wilhelmina H.A.M. Mulders The Auditory Laboratory, School of Anatomy, Physiology and Human Biology, M311, The University of Western Australia, 35 Stirling hwy, Crawley, Western Australia 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2011 Received in revised form 31 January 2012 Accepted 2 February 2012 Available online 13 February 2012
Hyperactivity in the form of increased spontaneous firing rates of single neurons develops in the guinea pig inferior colliculus (IC) after unilateral loud sound exposures that result in behavioural signs of tinnitus. The hyperactivity is found in those parts of the topographic frequency map in the IC where neurons possess characteristic frequencies (CFs) closely related to the region in the cochlea where lasting sensitivity changes occur as a result of the loud sound exposure. The observed hyperactivity could be endogenous to the IC, or it could be driven by hyperactivity at lower stages of the auditory pathway. In addition to the dorsal cochlear nucleus (DCN) hyperactivity reported by others, specific cell types in the ventral cochlear nucleus (VCN) also show hyperactivity in this animal model suggesting that increased drive from several regions of the lower brainstem could contribute to the observed hyperactivity in the midbrain. In addition, spontaneous afferent drive from the cochlea itself is necessary for the maintenance of hyperactivity up to about 8 weeks post cochlear trauma. After 8 weeks however, IC hyperactivity becomes less dependent on cochlear input, suggesting that central neurons transition from a state of hyperexcitability to a state in which they generate their own endogenous firing. The results suggest that there might be a “therapeutic window” for early-onset tinnitus, using treatments that reduce cochlear afferent firing. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Elevated levels of spontaneous neural firing (spontaneous hyperactivity) have been described in a number of brain regions after treatments that are known to induce tinnitus in humans and that cause behavioural changes consistent with tinnitus in animal experiments. Numerous animal models have shown a consistent pattern of hyperactivity in the inferior colliculus (IC) after unilateral partial deafness induced by acoustic trauma and other forms of damage (Bauer et al., 2008; Dong et al., 2009,b; Mulders and Robertson, 2009). This hyperactivity appears to be associated with altered patterns of gene expression in the IC, including reduced expression of elements of inhibitory, notably GABAergic synaptic machinery and of Kþ ion channels that stabilize membrane potentials (Dong et al., 2009, 2010a,b). Although Abbreviations: IC, inferior colliculus; VCN, ventral cochlear nucleus; DCN, dorsal cochlear nucleus; PL, primary-like; PLn, primary-like with notch; CF, characteristic frequency; CAP, compound action potential; GPIAS, gap-induced suppression of acoustic startle response; MOCS, medial olivocochlear system; PSTH, peristimulustime histogram; CNS, central nervous system; GABA, g amino butyric acid. * Corresponding author. Fax: þ61 8 6488 1025. E-mail addresses: [email protected], [email protected] (D. Robertson). 0378-5955/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2012.02.002
a causal relationship between IC hyperactivity and behavioural tinnitus has not been definitively established, the central role of this structure in the ascending auditory pathways means that midbrain hyperactivity continues to be used as a promising model for the neural substrates of this potentially debilitating disorder. An important question is to what extent the hyperactivity in the IC (and possibly therefore, that seen in even higher parts of the auditory pathway such as the auditory cortex) is a result of altered intrinsic properties of the IC neurons and their local circuitry, and how much is dependent on input from lower stages of the pathway, such as the cochlear nucleus and even the cochlea itself. There are numerous reports that cochlear trauma of the type that leads both to behavioural tinnitus and to midbrain hyperactivity, also produces hyperactivity in the dorsal subdivision of the cochlear nucleus (DCN) (Kaltenbach et al., 2000, 2004) but until recently, the ventral subdivision (VCN) had not been directly investigated. With regard to the role of the cochlea, little attention has been paid to the relationship between spontaneous cochlear neural activity, tinnitus and IC hyperactivity, aside from the fact that some loss of cochlear afferent drive to the CNS appears to be a common precipitating cause of tinnitus. Indeed there is a generally pervasive view that while cochlear trauma may initiate the chain of events that eventually leads to tinnitus and the associated abnormal central neural
D. Robertson et al. / Hearing Research 295 (2013) 124e129
activity, tinnitus itself is essentially a central phenomenon, independent of ongoing background afferent input from the cochlea for its maintenance. In this paper, we review some of our recent published findings on hyperactivity in the brainstem and midbrain and also present some new data. We seek to address several questions: 1) how precise is the relationship between changes in cochlear afferent neural sensitivity and the features of hyperactivity in the IC? 2) is the VCN also a potential source of abnormal input to the IC? and 3) how dependent is central neural hyperactivity on the presence of ongoing spontaneous firing of cochlear primary afferent neurons? 2. Methods For all the experiments reported here, detailed electrophysiological methods have already been published (Mulders and Robertson, 2009, 2011; Vogler et al., 2011). In brief, groups of anaesthetized pigmented guinea pigs were subjected to pure tone acoustic trauma in one ear. After recovery times ranging from 1 to 12 weeks, terminal experiments were performed, again under general anaesthesia. In the terminal experiments, microelectrode recordings were made from large numbers of single neurons (70e110 per animal) in the central nucleus of the IC contralateral to the traumatized cochlea, or from single neurons in the ipsilateral VCN. In the case of IC recordings, the depth at which each neuron was encountered was recorded, as well as characteristic frequency (CF) and spontaneous firing rate estimated from a 10 s sample in the absence of external acoustic stimulation. For cochlear nucleus recordings, CF and spontaneous firing rate were recorded for each neuron encountered, as well as peristimulus-time histograms (PSTHs) in order to enable classification of neuron response type. In both IC and VCN recordings, all the data discussed were obtained from true single neuron recordings and not, as is often the case in several other studies, from multiunit or “ensemble” activity. In some terminal experiments, the spontaneous neural activity emanating from the cochlea was acutely silenced using either complete cochlear ablation, or cochlear perfusion of drugs that blocked either neurotransmitter release from inner hair cells or the postsynaptic receptors responding to the inner hair cell neurotransmitter (Mulders and Robertson, 2009). It is important to stress that these methods of cochlear silencing were acute, not chronic, and were performed on animals after the recovery period from initial acoustic trauma had taken place. Hence in these experiments, we observed the immediate effects of such treatments on neural hyperactivity that had already developed in the IC. We have also studied the immediate effects of electrical stimulation of the medial olivocochlear system (MOCS) on IC hyperactivity (Mulders et al., 2010). This was achieved by delivering trains of shocks to the MOCS axons at the floor of the IVth ventricle, while continuously recording the spontaneous firing of individual hyperactive neurons in the IC. The contribution of the intracochlear action of the MOCS system to the effects seen was evaluated by simultaneous intracochlear perfusion of strychnine, a potent reversible blocker of MOCS action on outer hair cells. Behavioural evaluation of tinnitus was performed using the gap suppression of acoustic startle method (GPIAS) reported in detail by Yang et al. (2007). An important consideration with this technique is that only unilateral acoustic insults can be routinely employed to induce hyperactivity and tinnitus, since the test relies on the subject responding to a gap in relatively low level background noise. Reduced sensitivity binaurally could make the noise and gap less audible and so artifactually bias the results. In all animals therefore, normal thresholds in the untreated cochlea were evaluated by recording of the compound action potential (CAP) audiogram prior to the final terminal experiments.
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3. Results 3.1. Features of IC hyperactivity Our animal model consistently shows the presence of neuronal hyperactivity (increased mean spontaneous firing rates) in IC. This hyperactivity is not an immediate reaction to the cochlear trauma, and takes time to develop. It is not evident immediately after cochlear trauma (Mulders and Robertson, 2011) but is significant from 1 week afterwards. The level of hyperactivity increases further between 1 and 2 weeks post cochlear trauma and is then maintained for up to 12 weeks post trauma (the longest recovery period that we have used in these experiments). In this animal model, mean spontaneous firing rates are close to zero in sham operated animals and in animals in which it is measured immediately after cochlear trauma. Mean spontaneous firing rates then increase to average values of 8e12 spikes/s at 1e12 weeks post exposure. As has been reported in other brain regions, hyperactivity is not uniform across the neuronal population in any given CF region, with many neurons in IC still showing spontaneous firing rates that are not higher than normal. Instead, hyperactivity is manifested as greatly increased firing rates in a relatively small subset of neurons. One of the questions still to be answered in IC is in what manner the subset of hyperactive neurons is different from the majority of IC neurons that do not show hyperactivity. In this animal model, it can be shown that hyperactivity in the IC can be accompanied by behavioural evidence of tinnitus. Fig. 1 shows the results obtained using the GPIAS test in a separate group of animals (n ¼ 4) between 7 and 12 days after inducing a high frequency hearing loss. The results after trauma show a reduction in gap suppression of startle (in this instance the gap did not significantly reduce the startle response) when using bandpass background noise centred at 14 kHz, but not when the noise is centred at 8 kHz. These results strongly suggest that these animals experience tinnitus with a spectral quality above about 10 kHz. This result might be predicted from the previous electrophysiological data which consistently showed after trauma localized to the high frequency region of the cochlea, that hyperactivity in the IC is significant for neurons with CF’s above 10 kHz, but not below (Mulders and Robertson, 2009, 2011; Mulders et al., 2011). The result is consistent with, but does not prove, a causal relationship between hyperactivity and tinnitus. The relationship between the detailed features of the peripheral cochlear trauma and the eventual distribution of hyperactivity in the IC was investigated in more detail by using two different
Fig. 1. Results of behavioural demonstration of tinnitus in group of guinea pigs (n ¼ 4) tested before and 7e12 days after trauma to the high frequency region of the cochlea. Vertical axis, % gap-induced suppression of startle using bandpass noise centred at 8 kHz (open bars) and 10 kHz (solid bars). Error bars (S.E.M). Note greatly reduced gap suppression of startle for 10 kHz background noise and lack of change for 8 kHz background noise.
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traumatizing exposures (5 kHz and 10 kHz at 124 dB SPL for 2 h) in separate groups of animals. The peripheral damage was estimated by careful measurement of the cochlear compound action potential (CAP) threshold audiogram. Fig. 2A and B shows that these two exposures result in clearly different patterns of peripheral sensitivity change. Fig. 2C and D shows the detailed relationship between these CAP threshold changes and the CF distribution of spontaneous firing rates in the IC. The latter is displayed as a running average of the spontaneous firing rates of all individual neurons encountered as a function of their CF. Fig. 2E shows the distribution of the hyperactivity per frequency region. It is clear from these data, that there is a rather tight relationship between the “existence region” of hyperactivity in the tonotopic map of the IC, and the frequencies at which peripheral CAP thresholds have been affected by the prior noise exposure. 3.2. Hyperactivity in VCN Similar forms of cochlear trauma to that employed in our studies of IC hyperactivity have been shown by others to induce hyperactivity in the dorsal subdivision of cochlear nucleus (DCN) (Kaltenbach et al., 2000, 2004). Until recently, the ventral subdivision (VCN) had not been directly investigated in this manner, even though the VCN provides a major source of indirect input to the IC. Therefore we have used our animal model to record from large numbers of single neurons in the VCN (Vogler et al., 2011). Clear evidence of hyperactivity was obtained, with average spontaneous firing rates being increased by 104% relative to sham operated animals. Fig. 3 shows the distribution of spontaneous rates across the range of CFs encountered in VCN. Interestingly, hyperactivity in VCN was more widespread in the tonotopic domain than was seen in IC, with significant elevation of mean spontaneous firing rates being found both when neurons were grouped according to CFs below and above 10 kHz. In this study, in addition to CF and spontaneous firing rates, neuron response type was also determined and it was found that only onset and primary-like neurons showed spontaneous firing rates that were significantly greater than in sham operated animals of the same age. When primary-like neurons were further categorized into primary-like (PL) or primary-like with notch (PLn) (Winter and Palmer, 1990), the greatest increase in spontaneous firing rates was seen for the PLn category, but this increase was not significant, probably because of the relatively small sample sizes when neurons are placed in these sub-categories of response type. Similarly, although a significant increase in spontaneous rate was found when all types of onset neurons were pooled, the sample size was not sufficient to indicate whether this rise was confined to particular onset response sub-categories. 3.3. Dependence on cochlear afferent drive We have shown that hyperactivity in the IC in our guinea pig model can be abolished, or greatly reduced, by silencing the spontaneous firing of primary cochlear afferent neurons (Mulders and Robertson, 2009). This has been demonstrated in two ways. In an initial set of experiments, after sampling hyperactivity from a large number of IC neurons, the originally traumatized cochlea contralateral to the IC was acutely ablated, and in the same animals, a further sampling was undertaken. It was clearly demonstrated using this method, that the hyperactivity was no longer present after such acute cochlear ablation. The acute ablation technique could have suffered from a number of confounding factors, and hence, in a further set of experiments, we silenced the contralateral cochlea using local cochlear cooling and by using intracochlear
perfusion of blockers of afferent firing (kainic acid and cobalt) while continuously recording the spontaneous firing of single hyperactive IC neurons. These experiments confirmed the results of the cochlear ablation studies, showing that the hyperactivity in IC neurons required intact neural activity from the cochlea to be maintained. These experiments on cochlear silencing were initially performed after 2 weeks recovery. We have also investigated the dependence of IC hyperactivity on cochlear integrity at recovery times up to 12 weeks post acoustic trauma (Mulders and Robertson, 2011). In these studies, it was found that the hyperactivity could be abolished by acute cochlear ablation up to 6 weeks post acoustic trauma. However, from 8 to 12 weeks post acoustic trauma, although cochlear ablation did cause some reduction in hyperactivity in the IC, this was not statistically significant. Hence the dependence of IC hyperactivity on continuing input from the contralateral cochlea appears to decrease the longer the hyperactivity has been present. 3.4. Effect of MOCS stimulation We have also shown that electrical stimulation of the MOCS transiently suppresses hyperactivity in single hyperactive IC neurons (Mulders et al., 2010). Intracochlear perfusion of strychnine showed that in most hyperactive IC neurons, this MOCSmediated suppression was almost entirely attributable to the intracochlear action of the MOCS. In view of the known action of MOCS in suppressing spontaneous cochlear neural output, this result provides additional support for the previous findings that at least at early stages in its development, central hyperactivity is dependent on continued neural drive from the cochlea. 4. Discussion In the guinea pig model reported here, central hyperactivity is not present within the first few hours post cochlear trauma (the time taken to collect sufficient single neuron data immediately after a cochlear acoustic trauma). Clearly therefore the model cannot provide a neural basis for tinnitus of sudden onset, since it appears to involve a progressive change in central neural activity that is observed, after a recovery period of 1 week post cochlear trauma. This limitation aside however, the results on IC hyperactivity show several interesting features that may assist in understanding the development of some forms of tinnitus, and their possible treatment. First the single neuron data both in the IC and the VCN reveal that hyperactivity is found only in a subset of neurons in any particular location. It is worth noting that such a conclusion could not be readily obtained from multiunit and ensemble recordings. A similar basic finding has been found in other brain regions as well, notably in the DCN in which it has been reported that only fusiform cells receiving convergent somatosensory and auditory input exhibit hyperactivity after cochlear trauma (Shore, 2011; Shore et al., 2008). These results in general suggest that the tinnitus percept may be the result of abnormal activity in discrete neural channels. Our finding of hyperactivity in primary-like and onset neurons in VCN is also consistent with this notion and it is interesting to note that recent studies suggest that abnormal excitability of the spherical bushy cell pathway from VCN (neurons which show primary-like characteristics) may be a key neural feature associated with tinnitus (Melcher this volume). These results could have implications for possible treatments, since attempts to reduce overall neural excitability, or increase overall inhibitory activity, may lack the specificity required to target the particular neural populations involved.
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Fig. 2. A and B, Effects of two different noise exposure regimes (10 kHz (B) and 5 kHz (A) traumatizing tone, 124 dB SPL, 2 h duration) on cochlear neural thresholds 2 weeks post exposure. Data for 10 kHz exposure group published previously (Mulders et al., 2011). Vertical axis, change in cochlear compound action potential threshold (CAP threshold) compared to pre-exposure thresholds. Asterisks denote changes that are statistically significant compared to pre-exposure thresholds (n ¼ 4 animals in each group). Data for 5 kHz group derived from 437 neurons. C and D, Relationship between CAP threshold changes (solid circles and line with error bars) and single neuron spontaneous firing rates in IC (continuous solid line). Single neuron firing rates displayed as a running average (n ¼ 29) of pooled data from all animals in each group. Data for 10 kHz exposure group published previously (Mulders et al., 2011). E, Histogram of mean spontaneous firing rates in IC per CF region in sham (white bars), 5 kHz (grey bars) and 10 kHz (black bars) acoustic trauma groups. *p < 0.05; **p < 0.01; #p < 0.001.
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Fig. 3. Mean spontaneous firing rates of neurons in VCN in sham (A) and acoustic trauma (B) groups. Data pooled from all neuron response categories and divided into 2 kHz CF bands. Solid circles and lines show average change in cochlear CAP thresholds after 2 weeks recovery, relative to initial thresholds in the two groups.
The finding that the pattern of hyperactivity in the IC matches remarkably well to the pattern of neural sensitivity changes in the cochlea (Fig. 2) is also of interest. Although we have not made detailed behavioural evaluations of tinnitus to see whether the perceived tinnitus spectrum shifts according to the shift in distribution of hyperactivity in these same animals, the result is consistent with observations in humans that the perceived tinnitus spectrum closely matches the pattern of threshold changes in the behavioural audiogram (Norena et al., 2002) and this strengthens the case for a possible causal relationship between hyperactivity and tinnitus. It should be noted however, that a close correspondence between peripheral threshold change, tinnitus and hyperactivity could be co-incidental. It has been reported that acoustic trauma can cause selective degeneration of higher threshold primary afferent neurons (Kujawa and Liberman, 2009), which would leave cochlear threshold unaltered. The most novel aspect of our results is the fact that cochlear silencing causes an immediate reduction of hyperactivity in the IC. This finding challenges the prevailing view that tinnitus, although it
is most commonly triggered by a cochlear event such as acoustic trauma, is fundamentally a maladaptive form of central plasticity in which the resulting abnormal neural activity is endogenously generated in central pathways. There is indeed ample evidence in humans and animal models that tinnitus can persist in the absence of cochlear afferent input to the brainstem (Kaltenbach, 2011; Zacharek et al., 2002). How can this be reconciled with our findings? Our working hypothesis (Fig. 4) does not rely on any suggestion that primary afferent spontaneous activity is elevated. Instead, we have suggested that the first stage of maladaptive plasticity is a hyperexcitability of central neurons that renders them more likely to generate action potentials in response to any remaining spontaneous afferent drive from the cochlea. In this early phase, elimination of the cochlear input will immediately eliminate the central hyperactivity. Our results show however, that there is a second, later phase, which we have dubbed “progressive centralization” (Mulders and Robertson, 2011) in which cochlear silencing is no longer completely effective. We suggest that this is the result of the central neurons becoming so excitable that they generate their own endogenous spontaneous activity. It is interesting to speculate on the mechanisms and the therapeutic implications of such “progressive centralization”. It may be that the initial cochlear trauma sets in motion a prolonged sequence of cellular events that is fixed in its progression. Alternatively, the early phase of hyperactivity may itself contribute to further maladaptive changes that lead to eventual centralization (Fig. 4). If the latter is the case, then temporarily reducing primary afferent firing early in the development of tinnitus might act to arrest, or even reverse, the progression to the more centralized state. The exploitation of this “therapeutic window” might form the basis of novel approaches to the treatment of tinnitus of recent onset. It is worth noting that the midbrain hyperactivity described in this review shows some significant differences from the behaviour of neurons of the cat auditory cortex after similar acoustic trauma. Spontaneous firing rates in auditory cortical neurons are not increased immediately after trauma (similar to our own results in IC), but hyperactivity does develop within 2 h post trauma and this hyperactivity appears to be no greater after more extended recovery periods (Norena and Eggermont, 2003, 2006). It is not clear therefore to what extent cortical hyperactivity may be independent of midbrain hyperactivity, and the implications of this for the notion of a “therapeutic window” targeting midbrain hyperactivity will require further investigation. Acknowledgements This work was supported by grants to D. Robertson and W. Mulders from Action on Hearing Loss, The Neurotrauma Research Program and the Medical Health and Research Infrastructure Fund. C. Bester and D. Vogler are currently recipients of Australian Postgraduate Awards. The authors are grateful to R. Salvi for advice and encouragement, D. Stolzberg for generous technical assistance and donation of the GPIAS software and I. Winter and I.M. Lloyd for providing single neuron software (Neurosound) and microelectrodes. References
Fig. 4. Schematic representation of proposed stages of development of IC hyperactivity.
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