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Erratum to “Noise-induced changes of neuronal spontaneous activity in mice inferior colliculus brain slices”
Neuroscience Letters 374 (2005) 74–79
Erratum
Erratum to “Noise-induced changes of neuronal spontaneous activity in mice inferior colliculus brain slices”夽 Dietmar Basta∗ , Arne Ernst Department of Otolaryngology at UKB, Free University of Berlin, Warener Strasse 7, 12683 Berlin, Germany
Abstract The inferior colliculus (IC) in vivo is reportedly subject to a noise-induced decrease of GABA-related inhibitory synaptic transmission accompanied by an amplitude increase of auditory evoked responses, a widening of tuning curves and a higher neuronal discharge rate at suprathreshold levels. However, other in vivo experiments which demonstrated constant neuronal auditory thresholds or unchanged spontaneous activity in the IC after noise exposure did not confirm those findings. Perhaps this can be the result of complex noise-induced interactions between different central auditory structures. It was, therefore, the aim of the present study to investigate the effects of noise exposure on the spontaneous electrical activity of single neurons in a slice preparation of the isolated mouse IC. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun 10:1). After one week, auditory brainstem response (ABR) recordings and extracellular single-unit recordings from spontaneously active neurons within the IC slice were performed in noise-exposed and in normal hearing control mice. Noise-exposed animals showed a significant ABR threshold shift in the whole tested frequency range and a significant lower neuronal spontaneous activity in all investigated isofrequency laminae compared to controls. In both groups, the firing rate of 80% of IC neurons (approximately) increased significantly during the application of the GABAA receptor antagonist Bicucullin (10 M). The present findings demonstrate a noise-related modulation of spontaneous activity in the IC, which possibly contribute to the generation of noise-induced tinnitus and hearing loss. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Noise; Spontaneous activity; Auditory midbrain; Brain slice
Hearing loss is primarily associated with cochlear outer hair cell (OHC) loss and/or spiral ganglion cell lesion after exposure to noise. However, cochlear pathophysiology cannot fully explain the resulting audiological symptoms in humans, such as tinnitus, hyperacusis, and loudness recruitment. This is also illustrated by the fact that transection of the auditory nerve is unable to abolish tinnitus [8] and patients with a moderate to severe OHC loss with a subsequent reduced auditory nerve output show complete recruitment of loudness and hyperacusis [32]. Neuroanatomical and neurophysiological consequences of noise exposure have been investigated in a number of doi of original article:10.1016/j.neulet.2004.07.030. Corresponding author. Tel.: +49 30 5681 3104; fax: +49 30 5681 2903. E-mail address: [email protected] (D. Basta).
∗
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.11.002
in vivo animal models. Cochlear damage results in neuronal shrinkage, axonal pruning and transynaptic degeneration [9,18–20,28], formation of new synapses [2], and modifications in the distribution of ascending projections [17] in several parts of the auditory pathway. As electrophysiological characteristics, a decrease of the spontaneous activity [5] and the compound action potential of the auditory nerve [26] were reported. However, noise exposure can lead to an increase of neuronal spontaneous activity in the dorsal cochlear nucleus [10,12,13] and a decrease in the ventral cochlear nucleus [24]. Moreover, the dorsal cochlear nucleus is characterized by a decrease in inhibition [26] and an alteration of tonotopic map arrangement [11] after noise exposure. In the primary auditory cortex, a tonotopic reorganization was observed following noise exposure. It was accompanied by an increase in amplitude of auditory evoked
D. Basta, A. Ernst / Neuroscience Letters 374 (2005) 74–79
potentials and a minor increase of spontaneous activity in those regions with reorganization [14,21,27]. In the inferior colliculus (IC), the amplitudes of auditory evoked potentials were elevated after noise exposure. That was particularly true for those stimulus frequencies near the low-frequency edge of hearing loss [25,30]. The GABA release [3] and the level of glutamate decarboxylase (which catalyzes the synthesis of GABA from glutamate) were lowered after intense acoustic exposure [1]. These findings correspond to a widening of tuning curves and a higher discharge rate at suprathreshold levels in neurons with a characteristic frequency below the noise frequencies [31]. Other studies show an increase of GABAergic activity after intense sound exposure [4] or cochlear ablation [7]. Surprisingly, no changes of single unit auditory threshold or spontaneous activity of investigated neurons were reported [31]. The earlier stated in vivo studies investigated extensively the effect of noise on specific centers of auditory processing within the auditory pathway in the anesthetized animal. The contrary results could be caused by different noise-related interactions between the structures of the auditory pathway, which occurs in vivo. All auditory structures are influenced by the noise exposure and as such noise-induced changes of a single structure or unit cannot be elucidated. The in vitro brain slice method provides the possibility to investigate a single brain structure disconnected from afferent and efferent inputs of the auditory pathway. Further this method discloses the confounding factors associated with anesthesia and allows a fast change of the chemical milieu. It was, therefore, the aim of the present study to investigate the noise-induced changes of spontaneously active single neurons in inferior colliculus brain slices. Male or female mice (Mus musculus, NMRI strain) were used under the European Communities Council Directive and Institutional Animal Care Guidelines. All efforts were made to minimize pain or discomfort. At the age of 21 days, normally hearing mice with a positive Preyer’s reflex were noise-exposed (narrow band (9.5–10.5 kHz) continuous noise with a total level of 115 dB for 3 h) under anesthesia (Ketamin 100 mg/kg, Rompun 10 mg/kg) in a closed sound-proof chamber (0.8 m × 0.8 m × 0.8 m, minimal attenuation 60 dB). Anesthesia was controlled by a video camera placed in front of the head of the animals. Body temperature was maintained at a constant level (37 ◦ C) by an infrared lamp. Frequency specific auditory brainstem response (ABR) recordings [29] were performed after one week in noiseexposed animals and in controls as well under anesthesia. The ABR-recordings were carried out by using free field acoustic stimulation (National Instruments, Austin, USA) in a soundproof chamber (minimal attenuation 60 dB). Subdermal needle electrodes were placed at the vertex (active), the mastoid (reference) and the upper neck (ground). The body temperature was maintained by a heating pad. The brain response was pre-amplified (in the sound-proof chamber), amplified (100,000×) and filtered (bandpass 0.03–10 kHz) by a DBA-
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S system (WPI, Sarasota, USA) stored, averaged (500×) and analyzed with the software Madlab (Edinburgh, UK). Immediately after the animal woke up, it was decapitated and the brain was carefully removed. Using a vibrating microtome (Vibroslice 752, WPI, Aston, UK), two 300 m thick frontal slices (including the IC) were microdissected from the whole preparation. After 2 h of incubation (washout of residual anesthetics) in carbogenized (95% O2 –5% CO2 ) artificial cerebrospinal fluid (cACSF) at 35 ◦ C, one slice was transferred to a submerged-type recording chamber. The recording chamber was continuously perfused (4.5 ml/min) with warm (37 ◦ C) cACSF. The cACSF contained the following concentrations (in mM): 124 NaCl, 3 KCl, 1.25 NaH2 PO4 , 1.8 MgSO4 , 1.6 CaCl2 , 10 glucose, 26 NaHCO3 . The temperature of the bath solution was measured with a thermistor probe in the recording chamber and regulated within a small range by a temperature controller (npi-electronics). Action potentials from spontaneously active neurons within the central and external nucleus of the IC were extracellularly recorded with glass electrodes. The electrodes were pulled on a P87 horizontal puller (Sutter Instruments, Novato, CA, USA) and back-filled with a sodium chloride solution (154 mM). The resulting electrode resistance was approximately 5 M. The signal was amplified (10,000×) and filtered (0.1–3 kHz band pass) (WPI, Aston, UK), visualized on an oscilloscope, digitized by a 1401 Plus interface (Cambridge Electronic Design Ltd., Cambridge, UK) and stored in the Spike 2 software format (Cambridge Electronic Design Ltd., Cambridge, UK). Spikes were detected offline by using a template-matching technique within the software Spike 2. After establishing a stable recording, neuronal spontaneous activity was measured for one minute. The GABAA receptor antagonist Bicucullin (10 M) was added to the bath solution to evaluate the GABAergic influence on the spontaneous activity of the isolated IC. In the present case, the brain slice preparation was used for the investigation of one single neuron only. The recording position within the slice was determined visually by using a grid ocular. The rostrocaudal location was measured by a micromanipulator (Narishige, Japan) and the difference between the surface of the slice and the most caudal extention of the IC (determined during microdissection) was added to this value. Using these information, the best frequency of the actual neuron under study was estimated in accordance with a histological atlas [22]. ABR threshold shift and neuronal changes of spontaneous activity were tested by the t- or u-test. The percentage of neurons in the frequency groups was compared by the χ2 -test. In addition, the distribution of the percentage of neurons in all frequency groups were compared by the Kolmogoroff–Smirnoff test. The neuronal firing rate of each individual neuron before and during Bicucullin application were compared by the Wilcoxon-test. The average firing rate during Bicucullin application of noise-exposed animals were compared with the average firing rate of neurons before Bicucullin application and the average firing rate of the controls by
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D. Basta, A. Ernst / Neuroscience Letters 374 (2005) 74–79
Fig. 1. Auditory brainstem response thresholds at different frequencies of noise-exposed mice and controls (n = 11 per group). Solid line and filled squares indicates threshold shift after noise exposure (related to the right scale). Zero attenuation corresponds to 80 dB.
the Wilcoxon or t-test (α-corrected). The significance level for all statistical analyses was P < 0.05. The ABR recordings showed in all investigated frequencies a significant threshold (upward) shift in noise-exposed animals (n = 11) compared to controls (n = 11). The auditory threshold shift range was between 33 and 54 dB SPL. This effect was most clearly observed within the frequency range of 10–16 kHz (Fig. 1). The standard deviation of ABR thresholds was significantly lower in noise-exposed animals than in the control group. During the extracellular recordings, only regular firing neurons with a stable and continuous spontaneous activity were considered for the analysis. The number of excluded neurons were almost similar in both groups (e.g., two bursting neurons in the normal hearing group and three in the noise-exposed group). The morphological location and spontaneous activity of all investigated neurons is shown in Fig. 2. The distribution of spontaneous active neurons was not significantly different between the two experimental groups. The average spontaneous spike activity of control ICneurons (n = 127) was 6.0 ± 5.2 imp/s (imp = spike) with a significant decrease in the frequency range between 10 and 15 kHz (Fig. 3). The proportion of spontaneously active neurons was significantly higher in the frequency range between 10 and 25 kHz than in all other investigated frequency ranges (Fig. 3). In noise-exposed animals the average spontaneous activity (and the corresponding standard deviation) of ICneurons decreased significantly to 1.1 ± 0.8 imp/s (n = 126). Interestingly, the spontaneous activity decreased significantly in all the investigated frequency ranges up to 30 kHz (Fig. 4). The distribution of spontaneously active neurons in all the frequency ranges was not affected by the noise exposure (Fig. 5). In control and noise-exposed animals, the firing rate of approximately 80% of IC-neurons significantly increased during the application of the GABAA receptor antagonist Bicu-
Fig. 2. Location and spontaneous activity of all investigated neurons in inferior colliculus brain slices of normal (A) and noise-exposed (B) mice. The spontaneous activity measured at these points are indicated by a grey scale (CN, central nucleus; CU, cunciform nucleus; DMN, dorsomedial nucleus; DN, dorsal nucleus; LL, lateral lemniscus; EN, external nucleus; PAG, periaqueductal gray).
cullin (10 M). The typical neuronal response behavior is shown in Fig. 6. Bicucullin application did not affect the spontaneous activity of 20% of neurons studied. The average spontaneous activity increased significantly during Bicucullin application to 3.5 ± 2.5 imp/s (n = 26) in noise-exposed
D. Basta, A. Ernst / Neuroscience Letters 374 (2005) 74–79
Fig. 3. Average spontaneous activity and the proportion of all spontaneously active neurons (in percentage of all investigated neurons) at different isofrequency laminae in inferior colliculus brain slices of the normal hearing mouse. Asterisks indicate significant differences to all other isofrequency groups (P < 0.05).
Fig. 4. Spontaneous activity of inferior colliculus neurons in the isofrequency laminae of brain slice preparations of noise-exposed mouse and controls. Asterisks and diagonal lines indicates the experimental groups which are significantly different (P < 0.05).
Fig. 5. Percentage of spontaneous active inferior colliculus neurons (in percentage of all investigated neurons) in the isofrequency laminae of brain slices of noise-exposed mouse and controls.
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Fig. 6. Typical electrophysiological response of a Bicucullin-sensitive inferior colliculus neuron in a brain slice of a noise-exposed mouse (solid bar indicates time-period of 10 M Bicucullin application).
animals and to 9.1 ± 4.3 imp/s (n = 28) in controls. The average firing rate of neurons in noise-exposed animals during Bicucullin application was not significantly different to those without Bicucullin application in controls (Fig. 7). The present study demonstrates that noise exposure can alter the intrinsic neuronal spontaneous activity of the IC, and this could be detected in an isolated brain slice preparation. The noise-induced, non-frequency-specific decrease in neuronal firing rate very much corresponds with the recorded broad-band ABR threshold shift. The effects might be such explicit, because young mice were exposed to noise just after the onset of hearing. Earlier studies show that young animals are more susceptible to noise than are adult animals [15,23]. Noise-induced tonotopic reorganization has already been previously described for the dorsal cochlear nucleus [11] and the auditory cortex [27]. Thus, the same could hold true for those related structures of the auditory midbrain. However, a possible tonotopic reorganization is not likely to be related to the present results since the noise-induced reduction in spontaneous activity was not isofrequency-laminae-specific.
Fig. 7. Average firing rate of inferior colliculus neurons in brain slices of noise-exposed mice before and during Bicucullin application (10 M) and of untreated normal hearing controls. Asterisks and diagonal lines indicate the experimental groups which are significantly different (P < 0.05).
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Our data does not confirm the previous in vivo findings where no changes in spontaneous activity were found after noise exposure in the IC [31]. Neuronal spontaneous activity measured in vivo does not necessarily reflect the spontaneous activity of the structure under study. Without totally blocking all synaptic influences in vivo, the intrinsic neuronal activity of a structure will essentially be determined by the contacting synapses from other auditory structures, which are dynamically influenced in their functional status by the noise exposure itself. This seems to be the reason for the non-uniform electrophysiological, but clear morphological results along the auditory pathway after noise exposure [2,9,18–20,28]. There are several reports with those non-uniform electrophysiological results. Viz., spontaneous activity after noise exposure in vivo is decreased in the auditory nerve [5], increased in the dorsal cochlear nucleus [10,12,13], decreased in the ventral cochlear nucleus [24] and increased to a minor extent in the primary auditory cortex [14,21,27]. In the IC, ongoing spontaneous electrical ensemble activity decreased [6]. However, single unit spontaneous activity remains unchanged [31]. Previous studies have shown a decrease of GABAergic synaptic activity after noise exposure [1,3,16] which in turn could explain the overshoot of auditory evoked potential amplitudes, a widening of tuning curve tails and a higher discharge rate at suprathreshold levels in IC neurons [31]. However, in related investigations a strong increase in GABAgene expression, a slight increase in Glutamate-gene expression [7] and an increase of GABA concentration [4] could be observed in the IC after intense sound. These latter findings are in accordance with the results of the present study, where the spontaneous activity decreased after noise exposure and the neurons of the noise-exposed animals were able to increase their firing rate during the block of GABAA inhibitory activity to a level not significantly different from those of the control group. The significant reduction of local spontaneous activity of the IC, as demonstrated in the present study, is one of the most prominent electrophysiological findings of noiserelated changes in the auditory midbrain. This could be one major aspect which correlates with the central auditory pathophysiology and, thus, with the clinical sequelae of noise. The primary (peripheral) targets of noise-induced lesions are in the organ of corti. However, an impaired processing of auditory information in a central structure such as the IC could explain the wide variety of clinical findings in noiseinduced hearing loss, e.g., tinnitus, hyperacusis, impaired speech understanding.
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[2] C.G. Benson, J.S. Gross, S.K. Suneja, S.J. Potashner, Synaptophysin immunoreactivity in the cochlear nucleus after unilateral cochlear or ossicular removal, Synapse 25 (3) (1997) 243–257. [3] S.C.J. Bledsoe, S. Nagase, J.M. Miller, R.A. Altschuler, Deafnessinduced plasticity in the mature central auditory system, Neuroreport 7 (1) (1995) 225–229. [4] K. Chen, M.A. Godfrey, D.A. Godfrey, Effects of intense sound exposure on amino acid concentrations in rat cochlea, cochlear nucleus and inferior colliculus, in: Proceedings of the 27th Congress of the Association for Research in Otolaryngology, 2004, p. 115. [5] P. Dallos, D. Harris, O. Ozdamar, A. Ryan, Behavioral, compound action potential, and single unit thresholds: relationship in normal and abnormal ears, J. Acoust. Soc. Am. 64 (1) (1978) 151– 157. [6] G.M. Gerken, S.S. Saunders, R.E. Paul, Hypersensitivity to electrical stimulation of auditory nuclei follows hearing loss in cats, Hear. Res. 13 (1984) 249–259. [7] A.G. Holt, M. Asako, C.A. Lomax, M.I. Lomax, R.A. Altschuler, Cochlear ablation results in differentially expressed neurotransmitter related genes in the auditory brainstem, in: Proceedings of the 27th Congress of the Association for Research in Otolaryngology, 2004, p. 299. [8] J.W. House, D.E. Brackmann, Tinnitus: surgical treatment, Ciba Found. Symp. 85 (1981) 204–216. [9] M. Jean-Babtiste, D.K. Morest, Transneuronal changes of synaptic endings and nuclear chromatin in the trapezoid body following cochlear ablation in cats, J. Comp. Neurol. 162 (1975) 111– 134. [10] J.A. Kaltenbach, D.A. Godfrey, J.B. Neumann, D.L. McCaslin, C.E. Afman, J. Zhang, Changes in spontaneous activity in the dorsal cochlear nucleus following exposure to intense sound, Hear. Res. 124 (1998) 78–84. [11] J.A. Kaltenbach, C.R. Kaplan, Changes in the tonotopic map of the dorsal cochlear nucleus following induction of cochlear lesions by exposure to intense sound, Hear. Res. 59 (1992) 213–223. [12] J.A. Kaltenbach, D. McCaslin, Increase of spontaneous activity in the dorsal cochlear nucleus following exposure to high intensity sound: a possible neural correlate of tinnitus, Audit. Neurosci. 3 (1996) 57–78. [13] J.A. Kaltenbach, M.A. Zacharek, J. Zhang, S. Frederick, Activity in the dorsal cochlear nucleus of hamsters previously tested for tinnitus following intense tone exposure, Neurosci. Lett. 355 (1/2) (2004) 121–125. [14] H. Komiya, J.J. Eggermont, Spontaneous firing activity of cortical neurons in adult cats with reorganized tonotopic map following puretone trauma, Acta Otolaryngol. 120 (6) (2000) 750–756. [15] M. Lenoir, G.R. Bock, R. Pujol, Supra-normal susceptibility to acoustic trauma of the rat cochlea, J. Physiol. (Paris) 75 (5) (1979) 521–524. [16] J.C. Milbrandt, T.M. Holder, M.C. Wilson, R.J. Salvi, D.M. Caspary, GAD levels and muscimol binding in rat inferior colliculus following acoustic trauma, Hear. Res. 147 (1/2) (2000) 251–260. [17] D.R. Moore, Auditory brainstem of the ferret: long survival following cochlear removal progressively changes projections from the cochlear nucleus to the inferior colliculus, J. Comp. Neurol. 339 (1994) 301–310. [18] D.K. Morest, Degeneration in the brain following exposure to noise, in: R.P. Hamernik, D.S.R.J. Henderson (Eds.), New Perspectives on Noise-Induced Hearing Loss, Raven Press, New York, 1982, pp. 87–93. [19] D.K. Morest, B. Bohne, Noise-induced degeneration in the brain and representation of inner and outer hair cells, Hear. Res. 9 (2) (1983) 145–151. [20] D.K. Morest, J. Kim, B.A. Bohne, Neuronal and transneuronal degeneration of auditory axons in the brainstem after cochlear lesions in the chinchilla: cochleotopic and non-cochleotopic patterns, Hear. Res. 103 (1997) 151–168.
D. Basta, A. Ernst / Neuroscience Letters 374 (2005) 74–79 [21] J. Popelar, J.P. Erre, J.M. Aran, Y. Cazals, Plastic changes in ipsicontralateral differences of auditory cortex and inferior colliculus evoked potentials after injury to one ear in the adult guinea pig, Hear. Res. 72 (1/2) (1994) 125–134. [22] R. Romand, G. Ehret, Development of tonotopy in the inferior colliculus. I. Electrophysiological mapping in house mice, Dev. Brain Res. 54 (2) (1990) 221–234. [23] N. Rybalko, J. Syka, Susceptibility to noise exposure during postnatal development in rats, Hear. Res. 155 (1/2) (2001) 32–40. [24] R.J. Salvi, R.P. Hamernik, D. Henderson, Discharge patterns in the cochlear nucleus of the chinchilla following noise induced asymptotic threshold shift, Exp. Brain Res. 32 (3) (1978) 301–320. [25] R.J. Salvi, S.S. Saunders, M.A. Gratton, S. Arehole, N. Powers, Enhanced evoked response amplitudes in the inferior colliculus of the chinchilla following acoustic trauma, Hear. Res. 50 (1/2) (1990) 245–257. [26] R.J. Salvi, J. Wang, D. Ding, Auditory plasticity and hyperactivity following cochlear damage, Hear. Res. 147 (1/2) (2000) 261– 274.
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[27] S. Seki, J.J. Eggermont, Changes in spontaneous firing rate and neural synchrony in cat primary auditory cortex after localized toneinduced hearing loss, Hear. Res. 180 (1/2) (2003) 28–38. [28] K.C. Sie, E.W. Rubel, Rapid changes in protein synthesis and cell size in the cochlear nucleus following eighth nerve activity blockade or cochlea ablation, J. Comp. Neurol. 320 (4) (1992) 501–508. [29] P.M. Sprenkle, J. McGee, J.M. Bertoni, E.J. Walsh, Development of auditory brainstem responses (ABRs) in Tshr mutant mice derived from euthyroid and hypothyroid dams, J. Assoc. Res. Otolaryngol. 2 (4) (2001) 330–347. [30] W.S. Szczepaniak, A. Moller, Evidence of neuronal plasticity within the inferior colliculus after noise exposure: a study of evoked potentials in the rat, Electroencephalogr. Clin. Neurophysiol. 100 (2) (1996) 158–164. [31] J. Wang, R.J. Salvi, N. Powers, Plasticity of response properties of inferior colliculus neurons following acute cochlear damage, J. Neurophysiol. 75 (1) (1996) 171–183. [32] F.G. Zeng, C.W. Turner, Binaural loudness matches in unilaterally impaired listeners, Q. J. Exp. Psychol. 43A (1991) 565–583.
Erratum
Erratum to “Noise-induced changes of neuronal spontaneous activity in mice inferior colliculus brain slices”夽 Dietmar Basta∗ , Arne Ernst Department of Otolaryngology at UKB, Free University of Berlin, Warener Strasse 7, 12683 Berlin, Germany
Abstract The inferior colliculus (IC) in vivo is reportedly subject to a noise-induced decrease of GABA-related inhibitory synaptic transmission accompanied by an amplitude increase of auditory evoked responses, a widening of tuning curves and a higher neuronal discharge rate at suprathreshold levels. However, other in vivo experiments which demonstrated constant neuronal auditory thresholds or unchanged spontaneous activity in the IC after noise exposure did not confirm those findings. Perhaps this can be the result of complex noise-induced interactions between different central auditory structures. It was, therefore, the aim of the present study to investigate the effects of noise exposure on the spontaneous electrical activity of single neurons in a slice preparation of the isolated mouse IC. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun 10:1). After one week, auditory brainstem response (ABR) recordings and extracellular single-unit recordings from spontaneously active neurons within the IC slice were performed in noise-exposed and in normal hearing control mice. Noise-exposed animals showed a significant ABR threshold shift in the whole tested frequency range and a significant lower neuronal spontaneous activity in all investigated isofrequency laminae compared to controls. In both groups, the firing rate of 80% of IC neurons (approximately) increased significantly during the application of the GABAA receptor antagonist Bicucullin (10 M). The present findings demonstrate a noise-related modulation of spontaneous activity in the IC, which possibly contribute to the generation of noise-induced tinnitus and hearing loss. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Noise; Spontaneous activity; Auditory midbrain; Brain slice
Hearing loss is primarily associated with cochlear outer hair cell (OHC) loss and/or spiral ganglion cell lesion after exposure to noise. However, cochlear pathophysiology cannot fully explain the resulting audiological symptoms in humans, such as tinnitus, hyperacusis, and loudness recruitment. This is also illustrated by the fact that transection of the auditory nerve is unable to abolish tinnitus [8] and patients with a moderate to severe OHC loss with a subsequent reduced auditory nerve output show complete recruitment of loudness and hyperacusis [32]. Neuroanatomical and neurophysiological consequences of noise exposure have been investigated in a number of doi of original article:10.1016/j.neulet.2004.07.030. Corresponding author. Tel.: +49 30 5681 3104; fax: +49 30 5681 2903. E-mail address: [email protected] (D. Basta).
∗
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.11.002
in vivo animal models. Cochlear damage results in neuronal shrinkage, axonal pruning and transynaptic degeneration [9,18–20,28], formation of new synapses [2], and modifications in the distribution of ascending projections [17] in several parts of the auditory pathway. As electrophysiological characteristics, a decrease of the spontaneous activity [5] and the compound action potential of the auditory nerve [26] were reported. However, noise exposure can lead to an increase of neuronal spontaneous activity in the dorsal cochlear nucleus [10,12,13] and a decrease in the ventral cochlear nucleus [24]. Moreover, the dorsal cochlear nucleus is characterized by a decrease in inhibition [26] and an alteration of tonotopic map arrangement [11] after noise exposure. In the primary auditory cortex, a tonotopic reorganization was observed following noise exposure. It was accompanied by an increase in amplitude of auditory evoked
D. Basta, A. Ernst / Neuroscience Letters 374 (2005) 74–79
potentials and a minor increase of spontaneous activity in those regions with reorganization [14,21,27]. In the inferior colliculus (IC), the amplitudes of auditory evoked potentials were elevated after noise exposure. That was particularly true for those stimulus frequencies near the low-frequency edge of hearing loss [25,30]. The GABA release [3] and the level of glutamate decarboxylase (which catalyzes the synthesis of GABA from glutamate) were lowered after intense acoustic exposure [1]. These findings correspond to a widening of tuning curves and a higher discharge rate at suprathreshold levels in neurons with a characteristic frequency below the noise frequencies [31]. Other studies show an increase of GABAergic activity after intense sound exposure [4] or cochlear ablation [7]. Surprisingly, no changes of single unit auditory threshold or spontaneous activity of investigated neurons were reported [31]. The earlier stated in vivo studies investigated extensively the effect of noise on specific centers of auditory processing within the auditory pathway in the anesthetized animal. The contrary results could be caused by different noise-related interactions between the structures of the auditory pathway, which occurs in vivo. All auditory structures are influenced by the noise exposure and as such noise-induced changes of a single structure or unit cannot be elucidated. The in vitro brain slice method provides the possibility to investigate a single brain structure disconnected from afferent and efferent inputs of the auditory pathway. Further this method discloses the confounding factors associated with anesthesia and allows a fast change of the chemical milieu. It was, therefore, the aim of the present study to investigate the noise-induced changes of spontaneously active single neurons in inferior colliculus brain slices. Male or female mice (Mus musculus, NMRI strain) were used under the European Communities Council Directive and Institutional Animal Care Guidelines. All efforts were made to minimize pain or discomfort. At the age of 21 days, normally hearing mice with a positive Preyer’s reflex were noise-exposed (narrow band (9.5–10.5 kHz) continuous noise with a total level of 115 dB for 3 h) under anesthesia (Ketamin 100 mg/kg, Rompun 10 mg/kg) in a closed sound-proof chamber (0.8 m × 0.8 m × 0.8 m, minimal attenuation 60 dB). Anesthesia was controlled by a video camera placed in front of the head of the animals. Body temperature was maintained at a constant level (37 ◦ C) by an infrared lamp. Frequency specific auditory brainstem response (ABR) recordings [29] were performed after one week in noiseexposed animals and in controls as well under anesthesia. The ABR-recordings were carried out by using free field acoustic stimulation (National Instruments, Austin, USA) in a soundproof chamber (minimal attenuation 60 dB). Subdermal needle electrodes were placed at the vertex (active), the mastoid (reference) and the upper neck (ground). The body temperature was maintained by a heating pad. The brain response was pre-amplified (in the sound-proof chamber), amplified (100,000×) and filtered (bandpass 0.03–10 kHz) by a DBA-
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S system (WPI, Sarasota, USA) stored, averaged (500×) and analyzed with the software Madlab (Edinburgh, UK). Immediately after the animal woke up, it was decapitated and the brain was carefully removed. Using a vibrating microtome (Vibroslice 752, WPI, Aston, UK), two 300 m thick frontal slices (including the IC) were microdissected from the whole preparation. After 2 h of incubation (washout of residual anesthetics) in carbogenized (95% O2 –5% CO2 ) artificial cerebrospinal fluid (cACSF) at 35 ◦ C, one slice was transferred to a submerged-type recording chamber. The recording chamber was continuously perfused (4.5 ml/min) with warm (37 ◦ C) cACSF. The cACSF contained the following concentrations (in mM): 124 NaCl, 3 KCl, 1.25 NaH2 PO4 , 1.8 MgSO4 , 1.6 CaCl2 , 10 glucose, 26 NaHCO3 . The temperature of the bath solution was measured with a thermistor probe in the recording chamber and regulated within a small range by a temperature controller (npi-electronics). Action potentials from spontaneously active neurons within the central and external nucleus of the IC were extracellularly recorded with glass electrodes. The electrodes were pulled on a P87 horizontal puller (Sutter Instruments, Novato, CA, USA) and back-filled with a sodium chloride solution (154 mM). The resulting electrode resistance was approximately 5 M. The signal was amplified (10,000×) and filtered (0.1–3 kHz band pass) (WPI, Aston, UK), visualized on an oscilloscope, digitized by a 1401 Plus interface (Cambridge Electronic Design Ltd., Cambridge, UK) and stored in the Spike 2 software format (Cambridge Electronic Design Ltd., Cambridge, UK). Spikes were detected offline by using a template-matching technique within the software Spike 2. After establishing a stable recording, neuronal spontaneous activity was measured for one minute. The GABAA receptor antagonist Bicucullin (10 M) was added to the bath solution to evaluate the GABAergic influence on the spontaneous activity of the isolated IC. In the present case, the brain slice preparation was used for the investigation of one single neuron only. The recording position within the slice was determined visually by using a grid ocular. The rostrocaudal location was measured by a micromanipulator (Narishige, Japan) and the difference between the surface of the slice and the most caudal extention of the IC (determined during microdissection) was added to this value. Using these information, the best frequency of the actual neuron under study was estimated in accordance with a histological atlas [22]. ABR threshold shift and neuronal changes of spontaneous activity were tested by the t- or u-test. The percentage of neurons in the frequency groups was compared by the χ2 -test. In addition, the distribution of the percentage of neurons in all frequency groups were compared by the Kolmogoroff–Smirnoff test. The neuronal firing rate of each individual neuron before and during Bicucullin application were compared by the Wilcoxon-test. The average firing rate during Bicucullin application of noise-exposed animals were compared with the average firing rate of neurons before Bicucullin application and the average firing rate of the controls by
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Fig. 1. Auditory brainstem response thresholds at different frequencies of noise-exposed mice and controls (n = 11 per group). Solid line and filled squares indicates threshold shift after noise exposure (related to the right scale). Zero attenuation corresponds to 80 dB.
the Wilcoxon or t-test (α-corrected). The significance level for all statistical analyses was P < 0.05. The ABR recordings showed in all investigated frequencies a significant threshold (upward) shift in noise-exposed animals (n = 11) compared to controls (n = 11). The auditory threshold shift range was between 33 and 54 dB SPL. This effect was most clearly observed within the frequency range of 10–16 kHz (Fig. 1). The standard deviation of ABR thresholds was significantly lower in noise-exposed animals than in the control group. During the extracellular recordings, only regular firing neurons with a stable and continuous spontaneous activity were considered for the analysis. The number of excluded neurons were almost similar in both groups (e.g., two bursting neurons in the normal hearing group and three in the noise-exposed group). The morphological location and spontaneous activity of all investigated neurons is shown in Fig. 2. The distribution of spontaneous active neurons was not significantly different between the two experimental groups. The average spontaneous spike activity of control ICneurons (n = 127) was 6.0 ± 5.2 imp/s (imp = spike) with a significant decrease in the frequency range between 10 and 15 kHz (Fig. 3). The proportion of spontaneously active neurons was significantly higher in the frequency range between 10 and 25 kHz than in all other investigated frequency ranges (Fig. 3). In noise-exposed animals the average spontaneous activity (and the corresponding standard deviation) of ICneurons decreased significantly to 1.1 ± 0.8 imp/s (n = 126). Interestingly, the spontaneous activity decreased significantly in all the investigated frequency ranges up to 30 kHz (Fig. 4). The distribution of spontaneously active neurons in all the frequency ranges was not affected by the noise exposure (Fig. 5). In control and noise-exposed animals, the firing rate of approximately 80% of IC-neurons significantly increased during the application of the GABAA receptor antagonist Bicu-
Fig. 2. Location and spontaneous activity of all investigated neurons in inferior colliculus brain slices of normal (A) and noise-exposed (B) mice. The spontaneous activity measured at these points are indicated by a grey scale (CN, central nucleus; CU, cunciform nucleus; DMN, dorsomedial nucleus; DN, dorsal nucleus; LL, lateral lemniscus; EN, external nucleus; PAG, periaqueductal gray).
cullin (10 M). The typical neuronal response behavior is shown in Fig. 6. Bicucullin application did not affect the spontaneous activity of 20% of neurons studied. The average spontaneous activity increased significantly during Bicucullin application to 3.5 ± 2.5 imp/s (n = 26) in noise-exposed
D. Basta, A. Ernst / Neuroscience Letters 374 (2005) 74–79
Fig. 3. Average spontaneous activity and the proportion of all spontaneously active neurons (in percentage of all investigated neurons) at different isofrequency laminae in inferior colliculus brain slices of the normal hearing mouse. Asterisks indicate significant differences to all other isofrequency groups (P < 0.05).
Fig. 4. Spontaneous activity of inferior colliculus neurons in the isofrequency laminae of brain slice preparations of noise-exposed mouse and controls. Asterisks and diagonal lines indicates the experimental groups which are significantly different (P < 0.05).
Fig. 5. Percentage of spontaneous active inferior colliculus neurons (in percentage of all investigated neurons) in the isofrequency laminae of brain slices of noise-exposed mouse and controls.
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Fig. 6. Typical electrophysiological response of a Bicucullin-sensitive inferior colliculus neuron in a brain slice of a noise-exposed mouse (solid bar indicates time-period of 10 M Bicucullin application).
animals and to 9.1 ± 4.3 imp/s (n = 28) in controls. The average firing rate of neurons in noise-exposed animals during Bicucullin application was not significantly different to those without Bicucullin application in controls (Fig. 7). The present study demonstrates that noise exposure can alter the intrinsic neuronal spontaneous activity of the IC, and this could be detected in an isolated brain slice preparation. The noise-induced, non-frequency-specific decrease in neuronal firing rate very much corresponds with the recorded broad-band ABR threshold shift. The effects might be such explicit, because young mice were exposed to noise just after the onset of hearing. Earlier studies show that young animals are more susceptible to noise than are adult animals [15,23]. Noise-induced tonotopic reorganization has already been previously described for the dorsal cochlear nucleus [11] and the auditory cortex [27]. Thus, the same could hold true for those related structures of the auditory midbrain. However, a possible tonotopic reorganization is not likely to be related to the present results since the noise-induced reduction in spontaneous activity was not isofrequency-laminae-specific.
Fig. 7. Average firing rate of inferior colliculus neurons in brain slices of noise-exposed mice before and during Bicucullin application (10 M) and of untreated normal hearing controls. Asterisks and diagonal lines indicate the experimental groups which are significantly different (P < 0.05).
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Our data does not confirm the previous in vivo findings where no changes in spontaneous activity were found after noise exposure in the IC [31]. Neuronal spontaneous activity measured in vivo does not necessarily reflect the spontaneous activity of the structure under study. Without totally blocking all synaptic influences in vivo, the intrinsic neuronal activity of a structure will essentially be determined by the contacting synapses from other auditory structures, which are dynamically influenced in their functional status by the noise exposure itself. This seems to be the reason for the non-uniform electrophysiological, but clear morphological results along the auditory pathway after noise exposure [2,9,18–20,28]. There are several reports with those non-uniform electrophysiological results. Viz., spontaneous activity after noise exposure in vivo is decreased in the auditory nerve [5], increased in the dorsal cochlear nucleus [10,12,13], decreased in the ventral cochlear nucleus [24] and increased to a minor extent in the primary auditory cortex [14,21,27]. In the IC, ongoing spontaneous electrical ensemble activity decreased [6]. However, single unit spontaneous activity remains unchanged [31]. Previous studies have shown a decrease of GABAergic synaptic activity after noise exposure [1,3,16] which in turn could explain the overshoot of auditory evoked potential amplitudes, a widening of tuning curve tails and a higher discharge rate at suprathreshold levels in IC neurons [31]. However, in related investigations a strong increase in GABAgene expression, a slight increase in Glutamate-gene expression [7] and an increase of GABA concentration [4] could be observed in the IC after intense sound. These latter findings are in accordance with the results of the present study, where the spontaneous activity decreased after noise exposure and the neurons of the noise-exposed animals were able to increase their firing rate during the block of GABAA inhibitory activity to a level not significantly different from those of the control group. The significant reduction of local spontaneous activity of the IC, as demonstrated in the present study, is one of the most prominent electrophysiological findings of noiserelated changes in the auditory midbrain. This could be one major aspect which correlates with the central auditory pathophysiology and, thus, with the clinical sequelae of noise. The primary (peripheral) targets of noise-induced lesions are in the organ of corti. However, an impaired processing of auditory information in a central structure such as the IC could explain the wide variety of clinical findings in noiseinduced hearing loss, e.g., tinnitus, hyperacusis, impaired speech understanding.
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