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Changes in cochlear electrical stimulation induced Fos expression in the rat inferior colliculus following deafness
Hearing Research 147 (2000) 242^250 www.elsevier.com/locate/heares
Changes in cochlear electrical stimulation induced Fos expression in the rat inferior colliculus following deafness Shigeyo Nagase, Josef M. Miller, Jerome Dupont, Hyun Ho Lim, Kazuo Sato, Richard A. Altschuler * Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan, 1301 East Ann Street, Ann Arbor, MI 48109-0506, USA Received 22 September 1999; accepted 28 March 2000
Abstract Fos immunoreactive (IR) staining was used to examine changes in excitatory neuronal activity in the rat inferior colliculus (IC) between normal hearing and 21 day deaf rats evoked by basal or apical monopolar cochlear electrical stimulation. The location of evoked Fos IR neurons was consistent with expected tonotopic areas. The number of Fos IR cells increased as stimulation intensity increased in both normal and 21 day deaf animals. Stimulation at 1.5U threshold evoked fewer Fos IR cells in 21 day deafened animals compared to normal hearing animals. At 5U and above, however, significantly increased numbers of Fos IR neurons (in a larger grouping) were evoked in 21 day deafened animals compared to normal hearing animals. Another group of animals had 7 days of deafness followed by 14 days of chronic basal cochlear electrical stimulation. In this group basal monopolar stimulation at 5U evoked not only a greater number of Fos IR neurons, compared to normal hearing animals, but the location of their grouping was slightly shifted to a more dorso-lateral region in the contralateral IC, compared to the normal hearing and 21 day deaf groups. These observations indicate that both deafness and chronic electrical stimulation may alter central auditory processing. ß 2000 Elsevier Science B.V. All rights reserved. Key words: c-fos; Inferior colliculus ; Plasticity ; Cochlear implant; Electrical stimulation; Auditory brain stem
1. Introduction There is increasing evidence that deafness can induce changes in the mature mammalian central auditory system that can alter processing when stimulation is reintroduced through cochlear prostheses (e.g. Miller et al., 1991, 1996; Schwartz et al., 1993; Snyder et al., 1990). Morphological changes following deafness include a loss of spiral ganglion cells (Jyung et al., 1989 ; Leake and Hradek, 1988; Webster and Webster, 1981 ; Zappia and Altschuler, 1989), changes in cell size in the cochlear nucleus (Hultcrantz et al., 1991; Lesperance et al., 1995 ; Lustig et al., 1994; Moore, 1991; Pasic and Rubel, 1989; Rubel et al., 1990; Webster, 1988), changes in auditory nerve synapses (Gulley et al., 1978; Miller et al., 1991; Rees et al., 1985; Redd
* Corresponding author. Tel.: +1 (734) 764 6115; Fax: +1 (734) 764 0014; E-mail: [email protected]
et al., 2000), and changes in glia (Canady and Rubel, 1992). Neurochemical changes include changes in GABA (Bergman et al., 1989; Bledsoe et al., 1995; Dupont et al., 1992; Helfert et al., 1999 ; Milbrandt et al., 2000 ; Mossop et al., 2000 ; Potashner et al., 1985; Suneja et al., 1998a), changes in glycine (Altschuler et al., 1995 ; Bergman et al., 1989 ; Suneja et al., 1998a,b) and glycine receptors (Altschuler et al., 1997 ; Potashner et al., 2000 ; Sanes, 1994 ; Suneja et al., 1998a,b). Age related changes have also been reported (Caspary et al., 1999 ; Helfert et al., 1999; Milbrandt and Caspary, 1995 ; Milbrandt et al., 1996, 1997 ; Willot et al., 1997 ; Willot and Turner, 2000) which may be related to deafness. These changes, as well as changes in connections, may contribute to functional changes that have been reported with deafness (e.g. Irvine et al., 2000 ; Rajan et al., 1993 ; Rajan and Irvine, 1996 ; Salvi et al., 1996, 2000; Snyder et al., 2000) as well as changes that occur when chronic electrical stimulation is re-introduced in the deafened ear (e.g. Leake et al.,
0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 1 3 4 - 9
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2000 ; Miller et al., 1996; Snyder et al., 1990). Thus, deafness results in a decrease in evoked 2-deoxyglucose (2DG) uptake in cells of the inferior colliculus (IC) which can be reversed with chronic stimulation (Schwartz et al., 1993). Snyder et al. (1990) have reported a broadening of the representation of site speci¢c stimulation in the cochlea across cells of the IC following chronic stimulation of the deafened ear. This correlates with observations that when the cochlea is partially destroyed the cochleotopic representation of the remaining portion increases its representation in auditory cortex (Irvine et al., 2000; Rajan and Irvine, 1996 ; Rajan et al., 1993). At this time, the mechanisms which directly underlie the changes in responsiveness of central auditory system cells in deafened animals remain unknown, although there is increasing evidence for the involvement of GABA (Bledsoe et al., 1995, 1997 ; Caspary et al., 1999; Mossop et al., 2000; Willot and Turner, 2000). Expression of the Fos proto-oncogene (c-fos) has now been demonstrated as a useful marker of neural excitation within the central auditory system (e.g. Adams, 1995; Brown and Liu, 1995; Ehret and Fischer, 1991 ; Friauf, 1991; Rouiller et al., 1992; Sato et al., 1992, 1993) with the constraint that small cells often have a lower threshold for expression than larger neurons (Adams, 1995; Brown and Liu, 1995). The location of Fos immunoreactive (IR) neurons in the IC evoked by narrow band sound (Brown and Liu, 1995) matches the tonotopic organization shown in anatomical studies (e.g. Adams, 1979; Oliver, 1984, 1985, 1987). Fos IR staining in the central auditory pathways can also be evoked by cochlear electrical stimulation (Saito et al., 1999; Vischer et al., 1994; Zhang et al., 1996). Saito et al. (1999) showed the localization of electrically evoked Fos IR neurons in the IC correlated with tonotopic organization and the number of cells (and the size of their grouping) increased with increased stimulation intensity. If there are changes in central auditory processing as a consequence of deafness or of chronic electrical stimulation, this should then be re£ected in changes in the number and/or pattern of neurons expressing Fos after cochlear electrical stimulation. The present study, therefore, examined the in£uence of 21 days of deafness, with or without chronic electrical stimulation, on evoked Fos in the rat IC. 2. Materials and methods Male Sprague^Dawley rats (250^350 g) were used in these experiments under protocols reviewed and approved by the University of Michigan Committee for the Use and Care of Animals. All subjects were treated
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with care under appropriate NIH Guidelines for the Care and Use of Laboratory Animals, and meeting AAALAC requirements. Normal hearing and bilaterally deafened control animals were assessed without stimulation. Normal hearing and bilaterally deafened animals received an implant in the base or the apex of the cochlea for monopolar stimulation. Animals were assessed for Fos IR neurons induced by electrical stimulation. The normal hearing and 21 day deaf animals receiving basal stimulation were subdivided to receive stimulation at 1.5U, 5U or 10U threshold. Apical stimulation animals received 5U stimulation in both normal hearing and 21 day deaf animals. Each condition had three animals except for the two control conditions which had two animals. A ¢nal group of three animals was bilaterally deafened and implanted with a basal monopolar prosthesis. These animals received suprathreshold chronic stimulation starting 7 days after deafening, for 14 days, and were then assessed with 5U EABR threshold stimulation. 2.1. Surgical preparation Animals were anesthetized with ketamine (75 mg/kg) and xylazine (8 mg/kg). The cochlea was exposed via a post-auricular approach, using aseptic procedures. For animals in the deafened groups, both cochleae were slowly perfused over 3 min with 30 Wl of 10% neomycin sulfate through the round window membrane. This procedure has been shown to produce a rapid and profound hearing loss (e.g. P¢ngst et al., 1995). For acute stimulation groups, electrodes were implanted 16 days after drug administration, while in the chronic group, implantation was performed 5 days after drug administration. A single Te£on coated 3T platinum iridium wire was placed in the base or the apex of the cochlea. For basal monopolar stimulation the wire ended in a 250 Wm diameter ball-tip which was placed into scala tympani through a small hole anterior to the round window. For apical monopolar stimulation the wire ended in a 150 Wm ball, placed in a small hole in the otic capsule of the apical turn. A 5T Pt-Ir wire without Te£on insulation was used as an indi¡erent electrode, placed along the bone within the bulla. A stainless steel vertex epidural electrode was threaded into the skull midway between bregma and lambda for electrophysiological studies. Pre- and post-operatively click evoked ABR thresholds were recorded to assure normal hearing (at onset) in all subjects included in this study, and to later measure drug induced hearing loss. An immediate threshold shift of 50 dB following neomycin treatment de¢ned the minimum acceptable loss for the bilateral deafness groups in this study.
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2.2. Physiological recording procedures EABR was assessed in all animals under light anesthesia with ketamine and xylazine, 2 days after implantation as well as prior to terminal perfusion. For chronically stimulated animals, additional EABRs were recorded on days 8 and 14, during chronic stimulation, to monitor stimulation e¤cacy. Threshold was de¢ned as the minimum peak intensity of stimulation that evoked a visually detectable consistent waveform. ABR responses were recorded to alternating condensation and rarefaction clicks (computer generated 0.16 ms square pulses), averaging over 1024 responses. EABRs were evoked by 50 Ws constant current pulses of alternating polarity, averaging over 2048 sweeps. 2.3. Chronic stimulation In the chronic stimulation group, stimulation was provided starting 7 days after deafening, and continuing for 14 days (until day 20). Chronic electrical stimulation consisted of charge balanced, 50 Ws/phase, biphasic square pulses, delivered at 200 Wamp for 4 h/day (for the 14 days). Chronic stimuli were generated and delivered via battery powered wearable stimulators that left the animals free to move in their cages. 2.4. Stimulation for inducing Fos Stimulation for inducing Fos IR was given under ketamine, 21 days after the initial deafening. Chronic animals were stimulated at 5U threshold, while acute animals were stimulated at 1.5, 5 or 10U EABR threshold. This stimulation was provided within a sound deadened, double walled booth, by an isolated constant current stimulator for 90 min. Stimuli were 50 Ws/phase biphasic, charge balanced square waves delivered at 100 pulses/s. 2.5. Immunohistochemical procedures Thirty minutes after the end of stimulation, animals were heavily anesthetized with chloral hydrate and perfused through the heart with 300 ml of 0.1 M phosphate bu¡er (PB, pH 7.3) followed by 300^500 ml of 4% paraformaldehyde in 0.1 M PB. Brains were removed, post¢xed in the same ¢xative for 2 h at 4³C and then placed in 20% sucrose in PB overnight at 4³C. The brain stem was frozen onto a cryostat chuck and 50 Wm frontal sections were cut through the auditory brain stem using a Hacker Bright cryostat. Free£oating sections were pre-incubated for 60 min at room temperature with 2% normal goat serum in phosphate bu¡ered saline (PBS) containing 1% bovine serum albumin, 0.3% Triton X-100 (PBS-T). Sections were then
incubated for 48 h at 4³C with primary antibody to Fos protein (Oncogene Sciences) diluted 1:2000 in PBS-T with 2% normal goat serum. Sections were rinsed three times with PBS (15 min per wash) and incubated with biotinylated goat anti-rabbit (Vector Laboratories) for 90 min. After washing the sections three times in PBS, they were incubated with avidin biotin peroxidase complex (ABC) (Vector Laboratories) for 1 h, rinsed three times in PBS and placed in nickel intensi¢ed diaminobenzidine (Vector Laboratories) for 2^3 min with the addition of H2 O2 . Sections were lightly counterstained with 2% thionine to allow di¡erentiation of cell types. Tissue sections were mounted onto chrome-alum, gelatin-subbed slides. Each section was examined by light microscopy. Sections were assessed under bright ¢eld optics on a Leitz Dialux or Zeiss Axioskop photomicroscope. The location of IR cells also was mapped on representative sections using a camera lucida attachment. While changes and/or di¡erences were apparent in qualitative assessments, quantitative measures were also done in order to evaluate the extent of the changes. Five comparable sections were assessed in each animal, using the Metamorph (Universal Imaging Inc) Image Analysis System. Images were acquired with a Dage/ MTI Precision 81 video camera on a Leitz Dialux photomicroscope and digitized with a Matrox LC board using Metamorph software. Metamorph programs were used to automatically threshold and count Fos IR neurons. With the intensi¢ed reaction product there was a v 7U di¡erence in pixel intensity between lightly stained and moderately-heavily stained nuclei and only neurons with moderately-heavily stained nuclei were counted. The number of labeled neurons in the central nucleus in a section was divided by the area of the central nucleus in each section to generate the density of Fos IR neurons. To assess and compare the number of Fos IR neurons located in groupings or bands in the IC, a grouping was considered to be a spatially limited accumulation of labeled neurons in a pro¢le of the IC containing at least 4 neurons/10 000 micron2 . Statistical analysis between groups was performed using the Wilcoxon signed rank test. To assist in determining changes in location in chronically stimulated versus non-chronically stimulated animals, the centroid (de¢ned as the center of the band or grouping of Fos IR neurons weighted for density) of the grouping was also determined and compared between sections from comparable coronal planes, by overlying the sections. 3. Results In normal as well as in acute and chronically stimulated 21 day deafened animals, the electrically induced
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Fig. 1. Low power camera lucida drawings of representative sections through the rat IC, comparing typical patterns of Fos IR neurons induced by cochlear basal monopolar electrical stimulation in normal hearing (A^C) and 21 day deafened (D^F) animals at di¡erent stimulation intensities; 1.5U threshold (A and C), 5U threshold (B and E) and 10U threshold (C and F). The number of induced Fos IR neurons increased with increased stimulation intensities in both normal hearing and 21 day deafened animals. At 1.5U stimulation fewer Fos IR neurons were induced in 21 day deafened animals (D) than in normal hearing animals (A). At higher stimulation intensities more Fos IR neurons were induced in 21 day deafened animals (E, F) than in normal hearing animals (B, C). Bar = 1 mm. Asterisk (*) shows the side ipsilateral to the stimulated cochlea.
Fos IR staining was con¢ned to the nucleus of individual neurons. No neurons in the auditory brain stem showed Fos IR in the absence of electrical stimulation. The number of Fos IR neurons evoked by cochlear electrical stimulation in auditory brain stem nuclei of normal hearing and 21 day deaf rats increased as stimulation intensity increased (Fig. 1).
3.1. Normal hearing animals With 1.5U stimulation of the basal turn of the cochlea, Fos IR cells were seen in a ventro-medial grouping in the high frequency region of the contralateral IC (Fig. 1A), containing an average of 26 cells/section in the central nucleus (Fig. 2A). Fewer cells were observed
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in the ipsilateral IC without any obvious banding pattern (Fig. 1A). When stimulation was increased to 5U EABR threshold a larger grouping of IR neurons was seen in the high frequency region of the contralateral IC containing an average of 61 cells/section, with few or no IR cells in the ipsilateral IC (Figs. 1B and 2A). After 10U stimulation, there were large increases in the number of Fos IR cells ¢lling much of the contralateral IC, with an average of 123 cells/section (Figs. 1C and 2A). The labeling in the ipsilateral IC was still relatively low. Apical cochlear stimulation at 5U threshold resulted in two (sometimes three) bands of Fos IR neurons in the contralateral IC, in a more dorso-lateral position compared to basal cochlear stimulation, containing an average of 107 IR cells per section (Figs. 2B and 3B). 3.2. 21 Day deaf animals ^ acute Basal cochlear stimulation at 1.5U threshold induced very few Fos IR neurons in the IC of 21 day deafened animals (Figs. 1D and 2A) with an average of 18 Fos IR cells in the ventro-medial region. This is signi¢cantly less than where evoked in normal hearing animals with 1.5U stimulation where an average of 26 cells in a grouping or band was observed (Figs. 1A and 2A). At both 5U and 10U stimulation, however, an increased number of evoked Fos IR cells was seen in 21 day deafened animals compared to normal hearing animals (Figs. 1 and 2A). There was an approximately 33% increase in the ventro-medial grouping of Fos IR cells, induced at the 5U stimulation level in 21 day deaf animals compared to normal hearing animals, and an approximately 60% increase at the 10U stimulation level over that seen in normal hearing animals (Figs. 1 and 2A). Apical stimulation was only assessed at 5U threshold and showed an approximately 70% increase in the number of Fos IR cells in 21 day deaf compared to normal hearing animals (Figs. 2B and 3B,D) and with a broader localization throughout the IC. 3.3. 21 Day deaf animals ^ chronic stimulation Basal stimulation at 5U threshold in animals that had received chronic stimulation, demonstrated a signi¢cantly di¡erent pattern of Fos IR neurons in the contralateral IC from what was observed in the acute group of 21 day deaf animals (Fig. 4). While the number of Fos IR neurons induced by 5U basal monopolar stimulation increased similarly in both deafened groups, compared to normal hearing animals, there was a difference in their location. In animals that had received chronic stimulation, two bands or groupings of Fos IR neurons were seen, with the larger grouping of Fos IR
Fig. 2. A: Bar graphs with counts of Fos positive neurons in bands or groupings in the contralateral IC stimulated at 1.5, 5 and 10U EABR threshold comparing normal hearing and 21 day deafened animals. A signi¢cant decrease in deafened animals is seen at 1.5U threshold stimulation and signi¢cant increases in deafened animals at 5U and 10U threshold stimulation. Error bars show standard error of the mean. B: Bar graph with counts of Fos positive neurons in bands or groupings from contralateral central nucleus of the IC comparing basal and apical monopolar stimulation at 5U threshold in normal hearing and 21 day deafened animals. Signi¢cant increases in Fos IR neurons in 21 day deaf animals are seen. Error bars show standard error of the mean.
neurons situated more dorso-lateral (Fig. 4) than the band induced in 21 day deaf acute animals. This dorso-lateral shift could also be seen by comparing the centroid of these bands or groupings. Fos induction in chronically stimulated deafened animals, compared to normal hearing animals, therefore demonstrated not only an increase in the number and spread of Fos IR neurons, but a shift in the topographic location not seen with deafness in the absence of chronic stimulation.
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Fig. 3. Low power camera lucida drawings of representative sections through the rat IC, showing typical patterns of Fos IR neurons induced with basal monopolar (ba) or apical monopolar (ap) cochlear electrical stimulation at 5U threshold in normal hearing (Hr) and 21 day deaf (Deaf) rats. Basal monopolar stimulation in normal hearing rats (A) induced a band or grouping of Fos IR neurons in the ventro-medial, high frequency region of the contralateral nucleus of the IC while in 21 day deaf animals an increased number of Fos IR neurons was induced in the ventro-medial region (C). Apical monopolar stimulation in the normal hearing rat (B) induced two bands or groupings of Fos IR neurons in lateral and medial regions of IC, with numbers increasing and the groupings blending together in the 21 day deafened rat (D). Bar = 1 mm. Asterisk (*) is on the side ipsilateral to the stimulated cochlea.
4. Discussion Several changes with deafness have been demonstrated in the present study. First, at low basal monopolar stimulation levels (1.5U threshold), less induction of Fos IR neurons was observed in the IC after 21 days of deafness, compared to normal hearing animals. This is consistent with our previous study using 2DG meth-
ods, in which we found decreased 2DG uptake evoked by 100 Wamp of electrical activity after 4 or 9 weeks of deafness (Schwartz et al., 1993). At stimulation intensities at or above 5U threshold, deafness resulted in an increased number of evoked Fos IR cells over that seen in normal hearing animals. Several mechanisms could explain a deafness related increase in the number of induced Fos IR neurons. There
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Fig. 4. Low power camera lucida drawings of representative sections through the rat IC comparing typical patterns of Fos IR neurons induced by cochlear basal monopolar electrical stimulation at 5U threshold in normal hearing (A), 21 day deaf (B) and deafened rats with chronic stimulation from days 7^20 (C). The number of induced cells increases in acute deafened animals (B), with a position shift in deafened animals with chronic stimulation (C). Bar = 1 mm. Arrowhead points to the side ipsilateral to the stimulated cochlea.
could be changes in the cochlea (e.g. loss of cellular elements) that result in increased current spread, causing broader activation of the auditory nerve. There could be an increase in the amount of excitation in the brain stem, from sprouting and spread of excitatory ¢bers and terminals within a speci¢c region of its inputs. There could also be a decrease in inhibition in that speci¢c region or in regions from which it receives input. None of these hypothetical mechanisms are mutually exclusive and all may be occurring to some extent. Results from Bledsoe et al. (1995) indicate that reduced inhibition may be involved in the IC. These studies found a reduction in GABA in the IC after 30 days of deafness. A reduction in GABA IR staining in the IC has been found with deafness (Dupont et al., 1992) and aging (Helfert et al., 1999). Deafness related changes in GABA and glycine release and uptake (Suneja et al., 1998a) and deafness and age related changes in GABA and glycine composition and/or binding (Altschuler et al., 1997; Caspary et al., 1999; Milbrandt et al., 1996, 1997; Suneja et al., 1998b) have been observed throughout the auditory brain stem and an age related deafness change in glycine and glycine receptor binding has been reported in the cochlear nucleus (Willot et al., 1997). Bledsoe et al. (1995, 1997) found that in normal hearing animals electrical stimulation resulted in suppression of activity in 40% of units examined in the central nucleus of the IC. After 21 days of deafness this suppression decreased to 5%. These observations of decreased inhibition, associated with de-
creases in inhibitory transmitter, could explain why the present study found increased numbers and spread of Fos IR neurons in the IC evoked by 5U threshold stimulation in 21 day deafened animals. Another important result of our studies is that chronic stimulation induces an additional change in the induction of Fos IR neurons from those induced by just a short period of deafness. In chronically stimulated animals, in addition to a deafness related increase in the number of Fos IR neurons induced with 5U stimulation, the location of the induced Fos IR cells shifted, compared to both normal hearing animals and acutely stimulated deafened animals. This suggests that more complex plastic changes are occurring. Snyder et al. (1990) showed that the average area in the IC activated by stimulation with electrodes that had been chronically stimulated at 6 dB above EABR threshold was approximately twice that observed in non-stimulated deafened animals and prior normal animals. It is also interesting that Rajan and Irvine (1996) and Irvine et al. (2000) report that a restricted cochlear lesion in the basal turn of the adult cat led to expanded representation in the IC of the cochleotopic frequencies at the edge of the lesion. They suggested that this change could be a result of reorganization in the IC or of pathways to the IC. These studies would indicate that chronic stimulation (acoustic or electrical) in a deafened auditory system, limited to simple repetitive activation of a restricted population of eighth nerve ¢bers may lead to expansion of the central representa-
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tion of the stimulated structures. Our studies would suggest that both deafness related changes and changes as a result of chronic stimulation of the deafened auditory pathways are occurring. The end results in the latter case most likely represent some combination of the two in£uences. This is consistent with results of Willot and Turner (2000) which show plastic changes in the IC induced by auditory deprivation (caused by genetic hearing loss) in DBA mice which are facilitated by augmented acoustic stimulation. The present study found more Fos IR neurons in the IC evoked by apical than by basal monopolar stimulation in both normal and 21 day deaf groups. It may be that more of the auditory nerve is excited by the apically located versus the basally located electrode, however there may be other di¡erences between basal and apical cochlea (e.g. Altschuler et al., 1991; Pujol et al., 1980) which could provide a di¡erence in thresholds and response. The current studies suggest that processing of auditory information changes as a consequence of a period of time of deafness and from arti¢cial characteristics of cochlear prosthetic stimulation, which could have important consequences when stimulation is re-introduced either through cochlear prostheses, or perhaps in the future through regenerated hair cells. Acknowledgements We would like to thank Diane Prieskorn for helpful technical assistance. These studies were supported by NIH/NIDCD grants DC 00383 and DC 00274. References Adams, J.C., 1979. Ascending projections to the inferior colliculus. J. Comp. Neurol. 183, 519^538. Adams, J.C., 1995. Sound stimulation induces fos related antigen in cells with common morphological properties throughout the auditory brainstem. J. Comp. Neurol. 361, 645^668. Altschuler, R.A., Raphael, Y., Prosen, C.A., Dolan, D.F., Moody, D.B., 1991. Acoustic stimulation and overstimulation in the cochlea: A comparison between basal and apical cochlea. In: Dancer, A., Henderson, D., Salvi, R.J., Hamernik, R. (Eds.), Noise Induced Hearing Loss. Mosby Year Book, St. Louis. Altschuler, R.A., Raphael, Y., Dupont, J., Sato, K., Miller, J.M., 1995. Active mechanisms in the response of the auditory system to over or under stimulation. In: Flock, A., Ottoson, D., Ulfendahl, M. (Eds.), Active Hearing. Elsevier, Amsterdam, pp. 239^56. Altschuler, R.A., Sato, K., Dupont, J., Bonneau, J.M., Nakagawa, H., 1997. NMDA and glycine receptors in the auditory brain stem. Diversity and changes with deafness. In: Sykka, J. (Ed.), Acoustic Signal Processing in the Central Auditory System. Plenum, New York. Bergman, M., Staatz-Benson, C., Potashner, S.J., 1989. Amino acid uptake and release in the guinea pig cochlear nucleus after inferior colliculus ablation. Hear. Res. 42, 283^291.
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Bledsoe, S.C., Nagase, S., Miller, J.M., Altschuler, R.A., 1995. Deafness-induced plasticity in the mature central auditory system. Neuroreport 7, 225^229. Bledsoe, S.C., Nagase, S., Altschuler, R.A., Miller, J.M., 1997. Changes in the central auditory system with deafness and return of activity via a cochlear prostheses. In: Sykka, J. (Ed.), Acoustic Signal Processing in the Central Auditory System. Plenum, New York, pp. 513^528. Brown, M.C., Liu, T.S., 1995. Fos-like immunoreactivity in central auditory neurons of the mouse. J. Comp Neurol. 357, 85^97. Canady, K.S., Rubel, E.W., 1992. Rapid and reversible astrocytic reaction to a¡erent activity blockade in chick cochlear nucleus. J. Neurosci. 12, 1001^1009. Caspary, D.M., Holder, T.M., Hughes, L.F., Milbrandt, J.C., McKernan, R.M., Naritoku, D.K., 1999. Age-related changes in GABAa receptor subunit composition and function in the rat auditory system. Neuroscience 93, 307^312. Dupont, J., Zoli, M., Agnati, L.F., Aran, J.M., 1992. Morphofunctional changes in guinea pig brainstem auditory nuclei after peripheral dea¡erentation: A hypothesis about central mechanisms of tinnitus. In: Aran, J.M., Dauman, R. (Eds.), Tinnitus. Kluger, Amsterdam, pp. 195^205. Ehret, G., Fischer, R., 1991. Neuronal activity and tonotopy in the auditory system visualized by c-fos gene expression. Brain Res. 567, 350^354. Friauf, E., 1991. C-fos immunohistochemistry reveals no shift of tonotopic order in the central auditory system of developing rats. Soc. Neurosci. Abst. 17, 123. Gulley, R.L., Wenthold, R.J., Neises, G.R., 1978. Changes in the synapses of spiral ganglion cells in the rostral anteroventral cochlear nucleus of the waltzing guinea pig. Brain Res. 158, 279^294. Helfert, R.H., Sommer, T.J., Meeks, J., Hofstetter, P., Hughes, L.F., 1999. Age-related synaptic changes in the central nucleus of the inferior colliculus of Fischer-344 rats. J. Comp. Neurol. 406, 285^298. Hultcrantz, M., Snyder, R., Rebscher, S., Leake, P., 1991. E¡ects of neonatal deafening and chronic intracochlear electrical stimulation on the cochlear nucleus in cats. Hear. Res. 54, 272^280. Irvine, D.R.F., Rajan, R.M., McDermott, H.J., 2000. Injury-induced reorganization in the adult auditory system and its perceptual consequences. Hear. Res. 147, 188^199. Jyung, R.W., Miller, J.M., Cannon, S.C., 1989. Evaluation of eighth nerve integrity by the electrically evoked middle latency response. Arch. Otolaryngol. Head Neck Surg. 101, 670^682. Leake, P.A., Hradek, G.T., 1988. Cochlear pathology of long term neomycin induced deafness in cats. Hear. Res. 33, 11^34. Leake, P.A., Snyder, R.L., Rebscher, S.J., Moore, C.M., Vollmer, M., 2000. Plasticity in central representations in the inferior colliculus induced by chronic single- vs. two-channel electrical stimulation by a cochlear implant after neonatal deafness. Hear. Res., this issue. Lesperance, M.M., Helfert, R.H., Altschuler, R.A., 1995. Deafness induced cell size changes in rostal AVCN of the guinea pig. Hear. Res. 86, 77^81. Lustig, L.R., Leake, P.A., Snyder, R.L., Rebscher, S.J., 1994. Changes in the cat cochlear nucleus following neonatal deafening electrical stimulation. Hear. Res. 74, 29^37. Milbrandt, J.C., Albin, R.L., Turgeon, S.M., Caspary, D.M., 1996. GABAa receptor binding in the aging rat inferior colliculus. Neuroscience 73, 449^458. Milbrandt, J.C., Hunter, C., Caspary, D.M., 1997. Alterations of GABA receptor subunit mRNA levels in aging Fischer 344 rat inferior colliculus. J. Comp. Neurol. 379, 455^465. Milbrandt, J.C., Caspary, D.M., 1995. Age dependent reduction of 3H strychnine binding sites in the cochlear nucleus of the Fischer 344 rat. Neuroscience 67, 713^719. Milbrandt, J.C., Holder, T.M., Wilson, M.C., Caspary, D.M., 2000.
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GAD levels and muscimol binding in rat inferior colliculus following acoustic trauma. Hear. Res., this issue. Miller, J.M., Altschuler, R.A., Niparko, J.K., Hartshorn, D.O., Helfert, R.H., Moore, J.K., 1991. Deafness induced changes in the central nervous system and their reversibility and prevention. In: Dancer, Henderson, D., Salvi, R.J., Hamernik (Eds.), Noise Induced Hearing Loss. Mosby Year Book, St. Louis. Miller, J.M., Altschuler, R.A., Dupont, J., Lesperance, M., Tucci, D., 1996. Consequences of deafness and electrical stimulation on the auditory system. In: Salvi, R.J., Henderson, D., Fiorino, F., Colletti, V. (Eds.), Auditory Plasticity and Regeneration. Tieman Med Publishers, New York, pp. 378^391. Moore, D.R., 1991. Development and plasticity of the ferret auditory system. In: Altschuler, R.A., Bobbin, R.P., Clopton, B.M., Ho¡man, D.W. (Eds.), Neurobiology of Hearing: The Central Auditory System. Raven Press, New York, pp. 461^76. Mossop, J.E., Wilson, M.J., Caspary, D.M., Moore, D.R., 2000. Dowwn-regulation of inhibition following unilateral deafening. Hear. Res. 147, 183^187. Oliver, D.L., 1984. Dorsal cochlear nucleus projections to the inferior colliculus in the cat: A light and electron microscopic study. J. Comp. Neurol. 224, 155^172. Oliver, D.L., 1985. Quantitative analyses of axonal endings in the central nucleus of the inferior colliculus and distribution of 3Hlabeling after injections in the dorsal cochlear nucleus. J. Comp. Neurol. 237, 343^359. Oliver, D.L., 1987. Projections to the inferior colliculus from the anteroventral cochlear nucleus in the cat: possible substrates for binaural interaction. J. Comp. Neurol. 264, 24^46. Pasic, T.R., Rubel, E.W., 1989. Rapid changes in cochlear nucleus cell size following blockade of auditory nerve electrical activity in gerbils. J. Comp. Neurol. 283, 474^480. P¢ngst, B.E., Morris, D.J., Miller, A.L., 1995. E¡ects of electrode con¢guration on threshold functions for electrical stimulation of the cochlea. Hear. Res. 85, 76^84. Potashner, S.J., Lindberg, N., Morest, D.K., 1985. Uptake and release of gamma-aminobutyric acid in the guinea pig cochlear nucleus after axotomy of cochlear and centrifugal ¢bers. J. Neurochem. 45, 1558^1566. Potashner, S.J., Suneja, S.K., Benson, C.G., 2000. Altered glycinergic synaptic activities in guinea pig brain stem auditory nuclei after unilateral cochlear ablation. Hear. Res., this issue. Pujol, R., Carlier, E., Lenoir, M., 1980. Ontogenic approach to inner and outer hair cell function. Hear Res. 423, 30. Rajan, R., Irvine, D.R.F., 1996. Features of and boundary conditions for lesion induced reorganization of adult cortical maps. In: Salvi, R.J., Henderson, D., Fiorini, F., Colletti, V. (Eds.), Auditory System Plasticity and Regeneration. Thieme Medical Publishers, New York, pp. 224^237. Rajan, R., Irvine, D.R.F., Wise, L.Z., Heil, P., 1993. E¡ect of partial cochlear lesions in adult cats on the representation of lesioned and unlesioned cochleas in primary auditory cortex. J. Comp. Neurol. 338, 17^49. Redd, E.E., Pongstaporn, T., Ryugo, D.K., 2000. The e¡ects of congenital deafness on auditory nerve synapses and globular bushy cells in cats. Hear. Res., this issue. Rees, S., Guldner, F.H., Aitkin, L., 1985. Activity dependent plasticity of postsynaptic density structure in the ventral cochlear nucleus of the rat. Brain Res. 325, 370^374. Rouiller, E.M., Wan, X.S.T., Moret, V., Liang, F., 1992. Mapping of c-fos expression elicited tones stimulation in the auditory pathways of the rat, with emphasis on the cochlear nucleus. Neurosci. Lett. 144, 19^24. Rubel, E.W., Hyson, R.L., Durham, D., 1990. A¡erent regulation of neurons in brain stem auditory system. J. Neurobiol. 21, 169^196.
Saito, H., Miller, J.M., P¢ngst, B.E., Altschuler, R.A., 1999. Fos immunoreactivity in the auditory brain stem evoked by bipolar intracochlear electrical stimulation: E¡ects of current level and pulse duration. Neuroscience 91, 139^161. Salvi, R.J., Wang, J., Ding, D., 2000. Auditory plasticity and hyperactivity following cochlear damage. Hear. Res. 147, 261^274. Salvi, R.J., Wang, J., Powers, N., 1996. Rapid functional reorganization in the inferior colliculus and cochlear nucleus after acute cochlear damage. In: Salvi, R.J., Henderson, D., Fiorini, F., Colletti, V. (Eds.), Auditory System Plasticity and Regeneration. Thieme Medical Publishers, New York, pp. 275^296. Sanes, D.H., 1994. Glycine receptor distribution is dependent on excitatory and inhibition a¡erents in the gerbil LSO. Assoc. Res. Otolaryngol. Midwinter Meet. 17, 10. Sato, K., Houtani, T., Ueyama, T., Ikeda, M., Yamashita, T., Kumazawa, T., Sugimoto, T., 1992. Mapping of the cochlear nucleus subregions in the rat with neuronal Fos protein induced by acoustic stimulation with low tones. Neurosci. Lett. 142, 48^52. Sato, K., Houtani, T., Ueyama, T., Ikeda, M., Yamashita, T., Kumazawa, T., Sugimoto, T., 1993. Identi¢cation of rat brainstem sites with neuronal fos protein induced by acoustic stimulation with pure tones. Acta Otolaryngol. (Stockh.) Suppl. 500, 18^22. Schwartz, D.R., Schacht, J., Miller, J.M., Frey, K., Altschuler, R.A., 1993. Chronic electrical stimulation reverses deafness-related depression of electrically evoked 2-deoxyglucose activity in the guinea pig inferior colliculus. Hear. Res. 70, 463^477. Snyder, R.L., Rebscher, S.J., Cao, K., Leake, P.A., Kelly, K., 1990. Chronic intracochlear electrical stimulation in the neonatally deafened cat. I: Expansion of central representation. Hear. Res. 50, 7^ 34. Snyder, R.L., Sinex, D.G., McGee, J.D., Walsh, E.W., 2000. Acute spiral ganglion lesions change the tuning and tonotopic organization of cat inferior colliculus neurons. Hear. Res., this issue. Suneja, S.K., Potashner, S.J., Benson, C.G., 1998a. Plastic changes in glycine and GABA release and uptake in the adult brain auditory nuclei after unilateral middle ear ossicle removal and cochlear ablation. Exp. Neurol. 151, 273^288. Suneja, S.K., Benson, C.G., Potashner, S.J., 1998b. Glycine receptors in the adult guinea pig brain auditory nuclei: regulation after cochlear ablation. Exp. Neurol. 154, 473^488. Vischer, M.W., Hausler, R., Rouiller, E.M., 1994. Distribution of Fos-like immunoreactivity in the auditory pathway of the Sprague-Dawley rat elicited by cochlear electrical stimulation. Neurosci. Res. 19, 175^185. Webster, M., Webster, D.B., 1981. Spiral ganglion neuron loss following organ of Corti loss. A quantitative study. Brain Res. 212, 17^ 30. Webster, D.B., 1988. Conductive hearing loss a¡ects the growth of the cochlear nuclei over an extended period of time. Hear. Res. 32, 185^192. Willot, J.F., Milbrandt, J.C., Bross, L.S., Caspary, D.M., 1997. Glycine immunoreactivity and receptor binding in the cochlear nucleus of C57BL/6J and CBA/CaJ mice: e¡ects of cochlear impairment and aging. J. Comp. Neurol. 385, 405^414. Willot, J.F., Turner, J.G., 2000. Central plasticity: relationships among hearing loss, augmented acoustic stimulation, responses of neurons in the inferior colliculus, and prepulse inhibition. Hear. Res. 147, 275^281. Zappia, J.J., Altschuler, R.A., 1989. Evaluation of the e¡ect of ototopical neomycin on spiral ganglion cell density in the guinea pig. Hear. Res. 40, 29^38. Zhang, J.S., Haenggeli, C.A., Tempini, A., Vischer, M.W., Moret, V., Rouiller, E.M., 1996. Electrically induced Fos-like immunoreactivity in the auditory pathway of the rat: E¡ects of survival time, duration, and intensity of stimulation. Brain Res. Bull. 39, 75^82.
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Changes in cochlear electrical stimulation induced Fos expression in the rat inferior colliculus following deafness Shigeyo Nagase, Josef M. Miller, Jerome Dupont, Hyun Ho Lim, Kazuo Sato, Richard A. Altschuler * Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan, 1301 East Ann Street, Ann Arbor, MI 48109-0506, USA Received 22 September 1999; accepted 28 March 2000
Abstract Fos immunoreactive (IR) staining was used to examine changes in excitatory neuronal activity in the rat inferior colliculus (IC) between normal hearing and 21 day deaf rats evoked by basal or apical monopolar cochlear electrical stimulation. The location of evoked Fos IR neurons was consistent with expected tonotopic areas. The number of Fos IR cells increased as stimulation intensity increased in both normal and 21 day deaf animals. Stimulation at 1.5U threshold evoked fewer Fos IR cells in 21 day deafened animals compared to normal hearing animals. At 5U and above, however, significantly increased numbers of Fos IR neurons (in a larger grouping) were evoked in 21 day deafened animals compared to normal hearing animals. Another group of animals had 7 days of deafness followed by 14 days of chronic basal cochlear electrical stimulation. In this group basal monopolar stimulation at 5U evoked not only a greater number of Fos IR neurons, compared to normal hearing animals, but the location of their grouping was slightly shifted to a more dorso-lateral region in the contralateral IC, compared to the normal hearing and 21 day deaf groups. These observations indicate that both deafness and chronic electrical stimulation may alter central auditory processing. ß 2000 Elsevier Science B.V. All rights reserved. Key words: c-fos; Inferior colliculus ; Plasticity ; Cochlear implant; Electrical stimulation; Auditory brain stem
1. Introduction There is increasing evidence that deafness can induce changes in the mature mammalian central auditory system that can alter processing when stimulation is reintroduced through cochlear prostheses (e.g. Miller et al., 1991, 1996; Schwartz et al., 1993; Snyder et al., 1990). Morphological changes following deafness include a loss of spiral ganglion cells (Jyung et al., 1989 ; Leake and Hradek, 1988; Webster and Webster, 1981 ; Zappia and Altschuler, 1989), changes in cell size in the cochlear nucleus (Hultcrantz et al., 1991; Lesperance et al., 1995 ; Lustig et al., 1994; Moore, 1991; Pasic and Rubel, 1989; Rubel et al., 1990; Webster, 1988), changes in auditory nerve synapses (Gulley et al., 1978; Miller et al., 1991; Rees et al., 1985; Redd
* Corresponding author. Tel.: +1 (734) 764 6115; Fax: +1 (734) 764 0014; E-mail: [email protected]
et al., 2000), and changes in glia (Canady and Rubel, 1992). Neurochemical changes include changes in GABA (Bergman et al., 1989; Bledsoe et al., 1995; Dupont et al., 1992; Helfert et al., 1999 ; Milbrandt et al., 2000 ; Mossop et al., 2000 ; Potashner et al., 1985; Suneja et al., 1998a), changes in glycine (Altschuler et al., 1995 ; Bergman et al., 1989 ; Suneja et al., 1998a,b) and glycine receptors (Altschuler et al., 1997 ; Potashner et al., 2000 ; Sanes, 1994 ; Suneja et al., 1998a,b). Age related changes have also been reported (Caspary et al., 1999 ; Helfert et al., 1999; Milbrandt and Caspary, 1995 ; Milbrandt et al., 1996, 1997 ; Willot et al., 1997 ; Willot and Turner, 2000) which may be related to deafness. These changes, as well as changes in connections, may contribute to functional changes that have been reported with deafness (e.g. Irvine et al., 2000 ; Rajan et al., 1993 ; Rajan and Irvine, 1996 ; Salvi et al., 1996, 2000; Snyder et al., 2000) as well as changes that occur when chronic electrical stimulation is re-introduced in the deafened ear (e.g. Leake et al.,
0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 1 3 4 - 9
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2000 ; Miller et al., 1996; Snyder et al., 1990). Thus, deafness results in a decrease in evoked 2-deoxyglucose (2DG) uptake in cells of the inferior colliculus (IC) which can be reversed with chronic stimulation (Schwartz et al., 1993). Snyder et al. (1990) have reported a broadening of the representation of site speci¢c stimulation in the cochlea across cells of the IC following chronic stimulation of the deafened ear. This correlates with observations that when the cochlea is partially destroyed the cochleotopic representation of the remaining portion increases its representation in auditory cortex (Irvine et al., 2000; Rajan and Irvine, 1996 ; Rajan et al., 1993). At this time, the mechanisms which directly underlie the changes in responsiveness of central auditory system cells in deafened animals remain unknown, although there is increasing evidence for the involvement of GABA (Bledsoe et al., 1995, 1997 ; Caspary et al., 1999; Mossop et al., 2000; Willot and Turner, 2000). Expression of the Fos proto-oncogene (c-fos) has now been demonstrated as a useful marker of neural excitation within the central auditory system (e.g. Adams, 1995; Brown and Liu, 1995; Ehret and Fischer, 1991 ; Friauf, 1991; Rouiller et al., 1992; Sato et al., 1992, 1993) with the constraint that small cells often have a lower threshold for expression than larger neurons (Adams, 1995; Brown and Liu, 1995). The location of Fos immunoreactive (IR) neurons in the IC evoked by narrow band sound (Brown and Liu, 1995) matches the tonotopic organization shown in anatomical studies (e.g. Adams, 1979; Oliver, 1984, 1985, 1987). Fos IR staining in the central auditory pathways can also be evoked by cochlear electrical stimulation (Saito et al., 1999; Vischer et al., 1994; Zhang et al., 1996). Saito et al. (1999) showed the localization of electrically evoked Fos IR neurons in the IC correlated with tonotopic organization and the number of cells (and the size of their grouping) increased with increased stimulation intensity. If there are changes in central auditory processing as a consequence of deafness or of chronic electrical stimulation, this should then be re£ected in changes in the number and/or pattern of neurons expressing Fos after cochlear electrical stimulation. The present study, therefore, examined the in£uence of 21 days of deafness, with or without chronic electrical stimulation, on evoked Fos in the rat IC. 2. Materials and methods Male Sprague^Dawley rats (250^350 g) were used in these experiments under protocols reviewed and approved by the University of Michigan Committee for the Use and Care of Animals. All subjects were treated
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with care under appropriate NIH Guidelines for the Care and Use of Laboratory Animals, and meeting AAALAC requirements. Normal hearing and bilaterally deafened control animals were assessed without stimulation. Normal hearing and bilaterally deafened animals received an implant in the base or the apex of the cochlea for monopolar stimulation. Animals were assessed for Fos IR neurons induced by electrical stimulation. The normal hearing and 21 day deaf animals receiving basal stimulation were subdivided to receive stimulation at 1.5U, 5U or 10U threshold. Apical stimulation animals received 5U stimulation in both normal hearing and 21 day deaf animals. Each condition had three animals except for the two control conditions which had two animals. A ¢nal group of three animals was bilaterally deafened and implanted with a basal monopolar prosthesis. These animals received suprathreshold chronic stimulation starting 7 days after deafening, for 14 days, and were then assessed with 5U EABR threshold stimulation. 2.1. Surgical preparation Animals were anesthetized with ketamine (75 mg/kg) and xylazine (8 mg/kg). The cochlea was exposed via a post-auricular approach, using aseptic procedures. For animals in the deafened groups, both cochleae were slowly perfused over 3 min with 30 Wl of 10% neomycin sulfate through the round window membrane. This procedure has been shown to produce a rapid and profound hearing loss (e.g. P¢ngst et al., 1995). For acute stimulation groups, electrodes were implanted 16 days after drug administration, while in the chronic group, implantation was performed 5 days after drug administration. A single Te£on coated 3T platinum iridium wire was placed in the base or the apex of the cochlea. For basal monopolar stimulation the wire ended in a 250 Wm diameter ball-tip which was placed into scala tympani through a small hole anterior to the round window. For apical monopolar stimulation the wire ended in a 150 Wm ball, placed in a small hole in the otic capsule of the apical turn. A 5T Pt-Ir wire without Te£on insulation was used as an indi¡erent electrode, placed along the bone within the bulla. A stainless steel vertex epidural electrode was threaded into the skull midway between bregma and lambda for electrophysiological studies. Pre- and post-operatively click evoked ABR thresholds were recorded to assure normal hearing (at onset) in all subjects included in this study, and to later measure drug induced hearing loss. An immediate threshold shift of 50 dB following neomycin treatment de¢ned the minimum acceptable loss for the bilateral deafness groups in this study.
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2.2. Physiological recording procedures EABR was assessed in all animals under light anesthesia with ketamine and xylazine, 2 days after implantation as well as prior to terminal perfusion. For chronically stimulated animals, additional EABRs were recorded on days 8 and 14, during chronic stimulation, to monitor stimulation e¤cacy. Threshold was de¢ned as the minimum peak intensity of stimulation that evoked a visually detectable consistent waveform. ABR responses were recorded to alternating condensation and rarefaction clicks (computer generated 0.16 ms square pulses), averaging over 1024 responses. EABRs were evoked by 50 Ws constant current pulses of alternating polarity, averaging over 2048 sweeps. 2.3. Chronic stimulation In the chronic stimulation group, stimulation was provided starting 7 days after deafening, and continuing for 14 days (until day 20). Chronic electrical stimulation consisted of charge balanced, 50 Ws/phase, biphasic square pulses, delivered at 200 Wamp for 4 h/day (for the 14 days). Chronic stimuli were generated and delivered via battery powered wearable stimulators that left the animals free to move in their cages. 2.4. Stimulation for inducing Fos Stimulation for inducing Fos IR was given under ketamine, 21 days after the initial deafening. Chronic animals were stimulated at 5U threshold, while acute animals were stimulated at 1.5, 5 or 10U EABR threshold. This stimulation was provided within a sound deadened, double walled booth, by an isolated constant current stimulator for 90 min. Stimuli were 50 Ws/phase biphasic, charge balanced square waves delivered at 100 pulses/s. 2.5. Immunohistochemical procedures Thirty minutes after the end of stimulation, animals were heavily anesthetized with chloral hydrate and perfused through the heart with 300 ml of 0.1 M phosphate bu¡er (PB, pH 7.3) followed by 300^500 ml of 4% paraformaldehyde in 0.1 M PB. Brains were removed, post¢xed in the same ¢xative for 2 h at 4³C and then placed in 20% sucrose in PB overnight at 4³C. The brain stem was frozen onto a cryostat chuck and 50 Wm frontal sections were cut through the auditory brain stem using a Hacker Bright cryostat. Free£oating sections were pre-incubated for 60 min at room temperature with 2% normal goat serum in phosphate bu¡ered saline (PBS) containing 1% bovine serum albumin, 0.3% Triton X-100 (PBS-T). Sections were then
incubated for 48 h at 4³C with primary antibody to Fos protein (Oncogene Sciences) diluted 1:2000 in PBS-T with 2% normal goat serum. Sections were rinsed three times with PBS (15 min per wash) and incubated with biotinylated goat anti-rabbit (Vector Laboratories) for 90 min. After washing the sections three times in PBS, they were incubated with avidin biotin peroxidase complex (ABC) (Vector Laboratories) for 1 h, rinsed three times in PBS and placed in nickel intensi¢ed diaminobenzidine (Vector Laboratories) for 2^3 min with the addition of H2 O2 . Sections were lightly counterstained with 2% thionine to allow di¡erentiation of cell types. Tissue sections were mounted onto chrome-alum, gelatin-subbed slides. Each section was examined by light microscopy. Sections were assessed under bright ¢eld optics on a Leitz Dialux or Zeiss Axioskop photomicroscope. The location of IR cells also was mapped on representative sections using a camera lucida attachment. While changes and/or di¡erences were apparent in qualitative assessments, quantitative measures were also done in order to evaluate the extent of the changes. Five comparable sections were assessed in each animal, using the Metamorph (Universal Imaging Inc) Image Analysis System. Images were acquired with a Dage/ MTI Precision 81 video camera on a Leitz Dialux photomicroscope and digitized with a Matrox LC board using Metamorph software. Metamorph programs were used to automatically threshold and count Fos IR neurons. With the intensi¢ed reaction product there was a v 7U di¡erence in pixel intensity between lightly stained and moderately-heavily stained nuclei and only neurons with moderately-heavily stained nuclei were counted. The number of labeled neurons in the central nucleus in a section was divided by the area of the central nucleus in each section to generate the density of Fos IR neurons. To assess and compare the number of Fos IR neurons located in groupings or bands in the IC, a grouping was considered to be a spatially limited accumulation of labeled neurons in a pro¢le of the IC containing at least 4 neurons/10 000 micron2 . Statistical analysis between groups was performed using the Wilcoxon signed rank test. To assist in determining changes in location in chronically stimulated versus non-chronically stimulated animals, the centroid (de¢ned as the center of the band or grouping of Fos IR neurons weighted for density) of the grouping was also determined and compared between sections from comparable coronal planes, by overlying the sections. 3. Results In normal as well as in acute and chronically stimulated 21 day deafened animals, the electrically induced
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Fig. 1. Low power camera lucida drawings of representative sections through the rat IC, comparing typical patterns of Fos IR neurons induced by cochlear basal monopolar electrical stimulation in normal hearing (A^C) and 21 day deafened (D^F) animals at di¡erent stimulation intensities; 1.5U threshold (A and C), 5U threshold (B and E) and 10U threshold (C and F). The number of induced Fos IR neurons increased with increased stimulation intensities in both normal hearing and 21 day deafened animals. At 1.5U stimulation fewer Fos IR neurons were induced in 21 day deafened animals (D) than in normal hearing animals (A). At higher stimulation intensities more Fos IR neurons were induced in 21 day deafened animals (E, F) than in normal hearing animals (B, C). Bar = 1 mm. Asterisk (*) shows the side ipsilateral to the stimulated cochlea.
Fos IR staining was con¢ned to the nucleus of individual neurons. No neurons in the auditory brain stem showed Fos IR in the absence of electrical stimulation. The number of Fos IR neurons evoked by cochlear electrical stimulation in auditory brain stem nuclei of normal hearing and 21 day deaf rats increased as stimulation intensity increased (Fig. 1).
3.1. Normal hearing animals With 1.5U stimulation of the basal turn of the cochlea, Fos IR cells were seen in a ventro-medial grouping in the high frequency region of the contralateral IC (Fig. 1A), containing an average of 26 cells/section in the central nucleus (Fig. 2A). Fewer cells were observed
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in the ipsilateral IC without any obvious banding pattern (Fig. 1A). When stimulation was increased to 5U EABR threshold a larger grouping of IR neurons was seen in the high frequency region of the contralateral IC containing an average of 61 cells/section, with few or no IR cells in the ipsilateral IC (Figs. 1B and 2A). After 10U stimulation, there were large increases in the number of Fos IR cells ¢lling much of the contralateral IC, with an average of 123 cells/section (Figs. 1C and 2A). The labeling in the ipsilateral IC was still relatively low. Apical cochlear stimulation at 5U threshold resulted in two (sometimes three) bands of Fos IR neurons in the contralateral IC, in a more dorso-lateral position compared to basal cochlear stimulation, containing an average of 107 IR cells per section (Figs. 2B and 3B). 3.2. 21 Day deaf animals ^ acute Basal cochlear stimulation at 1.5U threshold induced very few Fos IR neurons in the IC of 21 day deafened animals (Figs. 1D and 2A) with an average of 18 Fos IR cells in the ventro-medial region. This is signi¢cantly less than where evoked in normal hearing animals with 1.5U stimulation where an average of 26 cells in a grouping or band was observed (Figs. 1A and 2A). At both 5U and 10U stimulation, however, an increased number of evoked Fos IR cells was seen in 21 day deafened animals compared to normal hearing animals (Figs. 1 and 2A). There was an approximately 33% increase in the ventro-medial grouping of Fos IR cells, induced at the 5U stimulation level in 21 day deaf animals compared to normal hearing animals, and an approximately 60% increase at the 10U stimulation level over that seen in normal hearing animals (Figs. 1 and 2A). Apical stimulation was only assessed at 5U threshold and showed an approximately 70% increase in the number of Fos IR cells in 21 day deaf compared to normal hearing animals (Figs. 2B and 3B,D) and with a broader localization throughout the IC. 3.3. 21 Day deaf animals ^ chronic stimulation Basal stimulation at 5U threshold in animals that had received chronic stimulation, demonstrated a signi¢cantly di¡erent pattern of Fos IR neurons in the contralateral IC from what was observed in the acute group of 21 day deaf animals (Fig. 4). While the number of Fos IR neurons induced by 5U basal monopolar stimulation increased similarly in both deafened groups, compared to normal hearing animals, there was a difference in their location. In animals that had received chronic stimulation, two bands or groupings of Fos IR neurons were seen, with the larger grouping of Fos IR
Fig. 2. A: Bar graphs with counts of Fos positive neurons in bands or groupings in the contralateral IC stimulated at 1.5, 5 and 10U EABR threshold comparing normal hearing and 21 day deafened animals. A signi¢cant decrease in deafened animals is seen at 1.5U threshold stimulation and signi¢cant increases in deafened animals at 5U and 10U threshold stimulation. Error bars show standard error of the mean. B: Bar graph with counts of Fos positive neurons in bands or groupings from contralateral central nucleus of the IC comparing basal and apical monopolar stimulation at 5U threshold in normal hearing and 21 day deafened animals. Signi¢cant increases in Fos IR neurons in 21 day deaf animals are seen. Error bars show standard error of the mean.
neurons situated more dorso-lateral (Fig. 4) than the band induced in 21 day deaf acute animals. This dorso-lateral shift could also be seen by comparing the centroid of these bands or groupings. Fos induction in chronically stimulated deafened animals, compared to normal hearing animals, therefore demonstrated not only an increase in the number and spread of Fos IR neurons, but a shift in the topographic location not seen with deafness in the absence of chronic stimulation.
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Fig. 3. Low power camera lucida drawings of representative sections through the rat IC, showing typical patterns of Fos IR neurons induced with basal monopolar (ba) or apical monopolar (ap) cochlear electrical stimulation at 5U threshold in normal hearing (Hr) and 21 day deaf (Deaf) rats. Basal monopolar stimulation in normal hearing rats (A) induced a band or grouping of Fos IR neurons in the ventro-medial, high frequency region of the contralateral nucleus of the IC while in 21 day deaf animals an increased number of Fos IR neurons was induced in the ventro-medial region (C). Apical monopolar stimulation in the normal hearing rat (B) induced two bands or groupings of Fos IR neurons in lateral and medial regions of IC, with numbers increasing and the groupings blending together in the 21 day deafened rat (D). Bar = 1 mm. Asterisk (*) is on the side ipsilateral to the stimulated cochlea.
4. Discussion Several changes with deafness have been demonstrated in the present study. First, at low basal monopolar stimulation levels (1.5U threshold), less induction of Fos IR neurons was observed in the IC after 21 days of deafness, compared to normal hearing animals. This is consistent with our previous study using 2DG meth-
ods, in which we found decreased 2DG uptake evoked by 100 Wamp of electrical activity after 4 or 9 weeks of deafness (Schwartz et al., 1993). At stimulation intensities at or above 5U threshold, deafness resulted in an increased number of evoked Fos IR cells over that seen in normal hearing animals. Several mechanisms could explain a deafness related increase in the number of induced Fos IR neurons. There
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Fig. 4. Low power camera lucida drawings of representative sections through the rat IC comparing typical patterns of Fos IR neurons induced by cochlear basal monopolar electrical stimulation at 5U threshold in normal hearing (A), 21 day deaf (B) and deafened rats with chronic stimulation from days 7^20 (C). The number of induced cells increases in acute deafened animals (B), with a position shift in deafened animals with chronic stimulation (C). Bar = 1 mm. Arrowhead points to the side ipsilateral to the stimulated cochlea.
could be changes in the cochlea (e.g. loss of cellular elements) that result in increased current spread, causing broader activation of the auditory nerve. There could be an increase in the amount of excitation in the brain stem, from sprouting and spread of excitatory ¢bers and terminals within a speci¢c region of its inputs. There could also be a decrease in inhibition in that speci¢c region or in regions from which it receives input. None of these hypothetical mechanisms are mutually exclusive and all may be occurring to some extent. Results from Bledsoe et al. (1995) indicate that reduced inhibition may be involved in the IC. These studies found a reduction in GABA in the IC after 30 days of deafness. A reduction in GABA IR staining in the IC has been found with deafness (Dupont et al., 1992) and aging (Helfert et al., 1999). Deafness related changes in GABA and glycine release and uptake (Suneja et al., 1998a) and deafness and age related changes in GABA and glycine composition and/or binding (Altschuler et al., 1997; Caspary et al., 1999; Milbrandt et al., 1996, 1997; Suneja et al., 1998b) have been observed throughout the auditory brain stem and an age related deafness change in glycine and glycine receptor binding has been reported in the cochlear nucleus (Willot et al., 1997). Bledsoe et al. (1995, 1997) found that in normal hearing animals electrical stimulation resulted in suppression of activity in 40% of units examined in the central nucleus of the IC. After 21 days of deafness this suppression decreased to 5%. These observations of decreased inhibition, associated with de-
creases in inhibitory transmitter, could explain why the present study found increased numbers and spread of Fos IR neurons in the IC evoked by 5U threshold stimulation in 21 day deafened animals. Another important result of our studies is that chronic stimulation induces an additional change in the induction of Fos IR neurons from those induced by just a short period of deafness. In chronically stimulated animals, in addition to a deafness related increase in the number of Fos IR neurons induced with 5U stimulation, the location of the induced Fos IR cells shifted, compared to both normal hearing animals and acutely stimulated deafened animals. This suggests that more complex plastic changes are occurring. Snyder et al. (1990) showed that the average area in the IC activated by stimulation with electrodes that had been chronically stimulated at 6 dB above EABR threshold was approximately twice that observed in non-stimulated deafened animals and prior normal animals. It is also interesting that Rajan and Irvine (1996) and Irvine et al. (2000) report that a restricted cochlear lesion in the basal turn of the adult cat led to expanded representation in the IC of the cochleotopic frequencies at the edge of the lesion. They suggested that this change could be a result of reorganization in the IC or of pathways to the IC. These studies would indicate that chronic stimulation (acoustic or electrical) in a deafened auditory system, limited to simple repetitive activation of a restricted population of eighth nerve ¢bers may lead to expansion of the central representa-
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tion of the stimulated structures. Our studies would suggest that both deafness related changes and changes as a result of chronic stimulation of the deafened auditory pathways are occurring. The end results in the latter case most likely represent some combination of the two in£uences. This is consistent with results of Willot and Turner (2000) which show plastic changes in the IC induced by auditory deprivation (caused by genetic hearing loss) in DBA mice which are facilitated by augmented acoustic stimulation. The present study found more Fos IR neurons in the IC evoked by apical than by basal monopolar stimulation in both normal and 21 day deaf groups. It may be that more of the auditory nerve is excited by the apically located versus the basally located electrode, however there may be other di¡erences between basal and apical cochlea (e.g. Altschuler et al., 1991; Pujol et al., 1980) which could provide a di¡erence in thresholds and response. The current studies suggest that processing of auditory information changes as a consequence of a period of time of deafness and from arti¢cial characteristics of cochlear prosthetic stimulation, which could have important consequences when stimulation is re-introduced either through cochlear prostheses, or perhaps in the future through regenerated hair cells. Acknowledgements We would like to thank Diane Prieskorn for helpful technical assistance. These studies were supported by NIH/NIDCD grants DC 00383 and DC 00274. References Adams, J.C., 1979. Ascending projections to the inferior colliculus. J. Comp. Neurol. 183, 519^538. Adams, J.C., 1995. Sound stimulation induces fos related antigen in cells with common morphological properties throughout the auditory brainstem. J. Comp. Neurol. 361, 645^668. Altschuler, R.A., Raphael, Y., Prosen, C.A., Dolan, D.F., Moody, D.B., 1991. Acoustic stimulation and overstimulation in the cochlea: A comparison between basal and apical cochlea. In: Dancer, A., Henderson, D., Salvi, R.J., Hamernik, R. (Eds.), Noise Induced Hearing Loss. Mosby Year Book, St. Louis. Altschuler, R.A., Raphael, Y., Dupont, J., Sato, K., Miller, J.M., 1995. Active mechanisms in the response of the auditory system to over or under stimulation. In: Flock, A., Ottoson, D., Ulfendahl, M. (Eds.), Active Hearing. Elsevier, Amsterdam, pp. 239^56. Altschuler, R.A., Sato, K., Dupont, J., Bonneau, J.M., Nakagawa, H., 1997. NMDA and glycine receptors in the auditory brain stem. Diversity and changes with deafness. In: Sykka, J. (Ed.), Acoustic Signal Processing in the Central Auditory System. Plenum, New York. Bergman, M., Staatz-Benson, C., Potashner, S.J., 1989. Amino acid uptake and release in the guinea pig cochlear nucleus after inferior colliculus ablation. Hear. Res. 42, 283^291.
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