Consequences of unilateral hearing loss: Time dependent regulation of protein synthesis in auditory brainstem nuclei

Hearing Research

Hearing Research 233 (2007) 124–134

www.elsevier.com/locate/heares

Research paper

Consequences of unilateral hearing loss: Time dependent regulation of protein synthesis in auditory brainstem nuclei K.A. Hutson a, D. Durham b, D.L. Tucci a

a,*

Department of Surgery, Division of Otolaryngology-Head and Neck Surgery, Duke University Medical Center, Box 3805, Durham, NC 27710, USA b Department of Otolaryngology, University of Kansas Medical Center, Kansas City, KS 66160, USA Received 19 June 2007; received in revised form 17 August 2007; accepted 23 August 2007 Available online 30 August 2007

Abstract Conductive hearing impairment results in marked changes in neuronal activity in the central auditory system, particularly in young animals [Tucci, D.L., Cant, N.B., Durham, D., 1999. Conductive hearing loss results in a decrease in central auditory system activity in the young gerbil. Laryngoscope 109, 1359–1371]. To better understand the effects of conductive hearing loss (CHL) on cellular metabolism, incorporation of 3H-leucine was used as a measure of protein synthesis in immature postnatal day 21 gerbils subjected to either unilateral CHL by malleus removal or profound sensorineural hearing loss by cochlear ablation. 3H-leucine uptake was measured after survival times of 6 or 48 h. Protein synthesis values were standardized to measurements from the abducens nucleus and compared with measurements from sham animals at similar age/survival times. Protein synthesis in the medial superior olive (MSO) was found to be significantly down-regulated (bilaterally) after CHL in animals surviving 48 h. However, 6 h after CHL manipulation, protein synthesis is up-regulated in MSO (bilaterally) and in the ipsilateral medial nucleus of the trapezoid body. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Leucine metabolism; Cochlear nucleus; Superior olivary nuclei; Cochlear ablation; Conductive hearing loss; Gerbil

1. Introduction Conductive hearing loss (CHL) changes the way sound is processed in the central auditory system. Using 2-deoxyglucose (2-DG) as a measure of neuronal activity, unilateral CHL has been shown to produce changes in glucose uptake, with significantly reduced uptake in the major afferent projection originating from the affected ear (Tucci et al., 1999). Effects are most marked in young, developing animals. In these animals, CHL results in a decrease that is statistically similar to that observed following cochlear Abbreviations: AVCN, anteroventral cochlear nucleus; CA, cochlear ablation; CHL, conductive hearing loss; contra, contralateral; ipsi, ipsilateral; LSO, lateral superior olive; LSOl, lateral limb of the lateral superior olive; LSOm, medial limb of the lateral superior olive; MSO, medial superior olive; MTB, medial nucleus of the trapezoid body; SH, sham * Corresponding author. Tel.: +1 919 684 6869; fax: +1 919 681 6881. E-mail address: [email protected] (D.L. Tucci). 0378-5955/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2007.08.003

ablation (CA), despite the more substantial (profound) hearing loss observed after the latter manipulation. Cochlear destruction causes transneuronal degeneration in central auditory pathways (e.g., Jean-Baptiste and Morest, 1975; Pasic et al., 1994; Morest and Bohne, 1997; Potashner et al., 1997; Tierney et al., 1997). Furthermore, studies of CA-induced plasticity within these pathways have shown that CA affects the internal metabolism of central auditory system neurons. For example, the regulation of glutamate and glycine release by protein kinase is altered by CA (Zhang et al., 2002, 2003a,b, 2004), as are signal transduction pathways (Suneja and Postashner, 2003) and cyclic AMP levels (Mo et al., 2006). CA also can induce the re-emergence of GAP-43 expression in adult animals (Illing et al., 1997; Michler and Illing, 2002; Kraus and Illing, 2004). Each of these findings suggests that CA may have an affect on central auditory system neurons at the gene level (Holt et al., 2005).

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Cellular changes following CHL are less dramatic (Webster and Webster, 1979; Webster, 1983a–c; Coleman and O’Connor, 1979; Blatchley et al., 1983; Trune and Morgan, 1988a,b; Doyle and Webster, 1991; Walsh and Webster, 1994; Tucci and Rubel, 1985; Tucci et al., 1987; Moore et al., 1989), but there is evidence that the auditory deprivation produced by CHL, particularly if unilateral, alters the way that sound is processed, at least in lower brainstem nuclei. Some authors suggest that the symmetry of input may be important in establishing and maintaining neural projections, and that unilateral CHL may alter the anatomical structure of bilaterally innervated nuclei (Killackey and Ryugo, 1977). Moore et al. (1989) found that, despite the lack of change in neuron area in ipsilateral CN following unilateral CHL, there was a significant change in the projection from the CN opposite the affected ear to the ipsilateral IC, reflecting a possible compensatory increase in input to the IC from the normal ear (uncrossed pathway). In the barn owl, Knudsen (1999) found evidence for altered localization cues and reorganization of binaurally innervated central auditory nuclei following unilateral CHL produced by occlusive earplug placement during a critical period in development. 2-DG uptake is decreased in the ipsilateral cochlear nucleus after unilateral CHL, and there is information from several studies indicating there may be up-regulation of the contralateral cochlear nucleus (Tucci et al., 1999). Following unilateral CHL in young adult (6 week old) gerbils, there is a slight but significant increase in 2-DG uptake in the contralateral cochlear nucleus that is not seen following cochlear ablation (Tucci et al., 1999). A similar pattern of change, although less marked, is observed in cytochrome oxidase (CO) activity following unilateral CHL (Tucci et al., 2001). In this experiment, for adult animals, a significant decrease in CO activity is observed in the ipsilateral and a significant increase is observed in the contralateral anteroventral cochlear nucleus (AVCN). Morphological changes have also been observed in the contralateral AVCN after unilateral hearing loss, where a slight increase in the size of spherical cells has been reported (e.g., Coleman and O’Connor, 1979; Dodson et al., 1994). Possible compensatory changes were also observed in the contralateral AVCN following CHL in adult guinea pigs (Sumner et al., 2005). In that study of binaural properties of AVCN neurons following unilateral conductive impairment, the investigators found a dramatic increase in the proportion of units in the ipsilateral AVCN that responded with excitation to broad band noise stimulation of the contralateral (intact) ear. One consequence of severe end organ damage on cellular metabolism is a decrease in protein synthesis in the ipsilateral cochlear nucleus (Steward and Rubel, 1985; Born and Rubel, 1988; Hyson and Rubel, 1989; Sie and Rubel, 1992). A similar decrease in protein synthesis has also been observed in the ipsilateral cochlear nucleus following unilateral CHL (Trune and Kiessling, 1988). However, little is known about how unilateral hearing loss affects protein

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synthesis in other auditory brainstem structures. In order to better understand some of the cellular events associated with changes in central auditory system activity subsequent to CHL, we initiated the current study to investigate protein synthesis in central auditory system nuclei in immature gerbils. 2. Materials and methods 2.1. Subjects Thirty-three Mongolian gerbils (Meriones unguiculatus) obtained from a commercial supplier (Charles River) were used in the present study. All anesthetic, operative, and postoperative procedures and care were approved by the Institutional Animal Care and Use Committee and followed NIH guidelines. All animals entered the experimental paradigm at postnatal day 21 (P21). Protein synthesis was examined in animals subsequent to a sham (SH), CHL, or CA procedure, with survival times of 6 h (SH = 5, CHL = 6, CA = 6) or 48 h (SH = 5, CHL = 7, CA = 4). Our nomenclature is a P21 animal that survived 6 h falls in the category of P21(21) meaning manipulation and assessment both on postnatal day 21; or P21(23) meaning manipulated on P21 and uptake assessed 48 h later on P23. Our choice of age was guided by past studies and by our interest in the effects of hearing loss in young animals beyond the age of hearing onset. Gerbils first respond to sound at approximately P12 (Finck et al., 1972; Woolf and Ryan, 1984; Ryan and Woolf, 1993). At P16, gerbils possess sufficient sensorimotor integration to accurately approach the source of a sound in space (Kelly and Potash, 1986). By P18 gerbils have mature middle and inner ears, and both the auditory nerve and ventral cochlear nucleus show adult-like physiological response characteristics (Woolf and Ryan, 1985). However, central auditory system development continues until at least P30 (Woolf and Ryan, 1985) and perhaps even longer (Poulsen et al., 2007). Thus gerbils at P21 have acoustic experience and a mature peripheral apparatus, yet central auditory system structures are continuing to develop. 2.2. Surgical procedures Animals were anesthetized with an IP injection of a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg). All surgical procedures were performed unilaterally on the left ear. Animals in the SH condition served as our anesthesia only control group. For CHL and CA animals, the fur was shaved behind the left ear, a postauricular incision was made and tissue surrounding external ear canal was dissected away, with care taken to preserve the exiting facial nerve. A small opening was made in the cartilaginous portion of the ear canal, and the tympanic membrane visualized. Using fine forceps, the tympanic membrane was punctured and the malleus gently removed. The stapes

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and oval window were then observed, and in no case was there evidence that malleus removal had compromised the oval window. For animals in the CHL condition, the surgical wound was then closed with cyanoacrylate glue. Animals in the CA condition underwent the same procedures as above, except that the promontory of the cochlea was visualized after malleus removal, fractured with forceps, and fluid suctioned from the cochlea until dry, and the wound closed. Previous measures of hearing impairment associated with these manipulations showed the malleus removal produces a loss of 33 to 57 dB with preserved cochlear integrity, whereas the CA produces a profound sensorineural hearing loss (Tucci et al., 1999). All animals were observed throughout recovery from anesthesia and were returned to their home cage, where they had free access to food and water.

was determined using bright-field illumination. All nuclei except the AVCN were sampled at approximately the 50% rostral-caudal level. The LSO was further divided into medial (LSOm) and lateral (LSOl) limbs (see Fig. 1C). For

2.3. Protein synthesis Protein synthesis was assessed using tritiated leucine incorporation over a 30-minute period just prior to the animals sacrifice (Steward and Rubel, 1985; Sie and Rubel, 1992). This short time period is adequate for amino acid incorporation onto protein, and limits active transport of the protein away from the cell body (see Steward and Rubel, 1985). After the appropriate post surgical survival time had elapsed (6 h or 48 h), each animal was briefly anesthetized with halothane and given an intramuscular injection of 3H-leucine (5 lCu/g; American Radiolabeled Chemicals, St. Louis, MO) diluted in sterile water (typically 0.1 cc injection for a 20 g animal). Thirty minutes later, animals were deeply anesthetized with sodium pentobarbital (150 mg/kg) and perfused transcardially with 10% phosphate buffered formalin. Heads were removed and postfixed for at least three days in the same fixative. Brains were removed, blocked, and placed in fresh fix for another 3 days, then dehydrated and embedded in paraffin. Serial sections (10 lm) of the brainstem were taken in the coronal plane and a 1:4 series mounted onto Fisher Superfrost Plus slides (Fisher Scientific, St. Louis, MO). Sections were deparaffinized, hydrated, dried, and coated with Kodak NTB-2 emulsion diluted 1:1 with distilled water. The slides were stored in light-proof containers at 2 °C, for 10 weeks. Slides were developed in Kodak D19, washed in distilled water, fixed in Kodak Rapid Fix, lightly counterstained with thionin, dehydrated and coverslipped with DPX (BDH, Poole, England). 2.4. Data analysis The central auditory system nuclei examined in all conditions were the anteroventral cochlear nucleus (AVCN), the medial nucleus of the trapezoid body (MTB), and the medial superior olive (MSO). In the P21(21) condition, the lateral superior olive (LSO) was also examined. Examples of the nuclei studied are shown in Fig. 1. For each brain, the rostral-caudal extent of each auditory nucleus

Fig. 1. Sections through the auditory brainstem from a P21(21) CHL animal illustrating the regions sampled in this study. (A) Low power photomicrograph of AVCN at approximately the 20% rostral-caudal level. This was the level chosen to sample from each brain. Scale bar (bottom right) = 500 lm. (B) High power photomicrograph showing tritiated leucine incorporation (silver grains) in the spherical cell region of AVCN. Scale bar = 50 lm. (C) Low power photograph of the superior olivary complex at approximately the 50% rostral-caudal level. LSO was divided into medial and lateral limbs by drawing a line down from the dorsal hilus (solid line). Scale bar = 500 lm. Abbreviations: AVCN, anterior ventral cochear nucleus; LSOm, medial limb of the lateral superior olive; LSOl, lateral limb of the lateral superior olive; MSO, medial superior olive; MTB, medial nucleus of the trapezoid body.

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comparisons were made using the Mann–Whitney U test (see Tucci et al., 2001). 3. Results We used cellular incorporation of 3H-leucine as a tool to measure the relative level of protein synthesis in central auditory system structures subsequent to a unilateral hearing loss induced by middle or inner ear manipulation. The incorporation of amino acid was measured over a brief 30minute ‘‘window’’ immediately preceding euthanasia. We found no significant left–right differences among SH animals of a particular age group. Therefore, left and right SH data for each structure were combined for further comparison. No significant left–right differences were found for abducens neurons in experimental animals of any age group, indicating that our surgical manipulations had no effect on the abducens nuclei. In the paragraphs that follow, all differences reported as significant had P values of at least P < 0.05. 3.1. Protein synthesis and cell area in sham animals The protein synthesis and cell area results from SH animals are shown in Fig. 2. Sham animals provide a picture of the relative level of protein synthesis at each sample ‘‘window’’ period, that is, leucine uptake in central auditory system nuclei as compared to the uptake level in the

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the AVCN, sections were chosen at approximately the rostral 20% level, the region populated predominantly by large spherical bushy cells (Cant and Morest, 1984). Approximately 50 cells were sampled from each auditory nucleus, on both the right and left sides of the brain, under 63X oil magnification. Tissue sections were analyzed by observers blind to the experimental conditions. The cellular incorporation of 3H-leucine was measured in auditory nuclei and standardized against uptake by a ‘‘neutral’’ structure (the abducens nucleus) in order to obtain a relative measure of incorporation level comparable across conditions. Variation in tissue staining between age groups necessitated that incorporation be measured in one of two ways. For the P21(21) animals, we measured uptake in terms of fractional density (area occupied by silver grains/cell area). In the P21(23) animals silver grains were counted manually and uptake was measured in terms of the number of silver grains over a cell. Sections were analyzed using a Zeiss Axioskop microscope. After regions of the auditory nuclei to be sampled were identified, images of the sections were scanned (MTI digital camera), sent to a Power Macintosh G3 computer and displayed on a color monitor. Individual cells were identified by the presence of a darkly stained cytoplasm and clear nucleus. The border of each cell was outlined on the screen and NIH Image software was used to determine the area of each cell. Then a grain value was determined for each cell (fractional density of silver grains, or number of grains over a cell). To correct for between animal variation in absolute levels of leucine incorporation, grain values of motorneurons in the abducens nuclei (25 neurons on each side of the brain) were measured in each animal. Abducens corrected values for each auditory structure were analyzed within each age and hearing loss condition. Abducens corrections (ABD corr): Mean grain values for neurons in each auditory structure were divided by the mean grain value of the abducens nucleus neurons of the same side of the brain. Thus, ABD corr = mean grain value of a structure/mean grain value of the ipsilateral abducens nucleus. This measure corrects for individual differences in absolute incorporation levels, and provides a measure of protein synthesis in auditory nuclei relative to a standard structure at all time periods. As a relative measure, the ABD corr values can be compared within each age condition of the experiment despite the different methods used for obtaining grain values. Mean cell areas for each auditory structure were standardized in the same manner to the mean cell area of the ipsilateral abducens nucleus. Given the relatively small number of animals in each condition of this study, and the proportional nature of our data, ABD corr values were subjected to non-parametric analysis procedures using STAT View software. First, Kruskal–Wallace tests were performed grouping animals by age and manipulation condition, and by ipsilateral and contralateral sides of the brain. For comparisons that were statistically significant (P < 0.05), post-hoc paired

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Fig. 2. Abducens corrected (ABD corr) leucine incorporation values (top) and ABD corr cell areas (bottom) for sham animals from each condition in this study. Bars represent the average corrected value for each structure, error bars = one standard error of the mean. Using the uptake of trititiated leucine in the abducens nucleus and the cell area of abducens neurons to standardize incorporation levels and cell areas in the auditory nuclei, reduces variation due to tissue processing and analysis procedures between groups. Note that there was a significant difference in MSO cell area between P21 and P23 (represented by the numerals 1).

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leucine incorporation than CA animals. Compared to SH animals, unilateral CHL significantly increased protein synthesis in MSO (bilaterally) and ipsilateral MTB. Fig. 3 also shows that there were no significant differences in protein synthesis for CA animals as compared to SH animals at this short time period. P21(21) cell area: Compared to SH animals, neither the CHL or CA manipulations resulted in a significant change in cell area. P21(23) synthesis: As seen in Fig. 4, animals manipulated on P21 and examined on P23 (after a 48 h survival) show a pattern of protein synthesis different from P21(21) animals, in that protein synthesis in both CHL and CA animals is lower than that in SH animals for all structures. However, only the bilateral decrease in synthesis in MSO of CHL animals was significantly different from SH. P21(23) cell area: CHL animals. Compared to SH, a significant decrease in cell area was observed in the ipsilateral AVCN, and ipsilateral MTB. Furthermore, there were significant differences in cell area between sides of the brain for AVCN and MTB, where cells ipsilateral to the manipulation were smaller than cells on the contralateral side. CA animals: As with CHL animals in this age group, there was a significant difference in cell area between ipsilateral and contralateral AVCN (ipsilateral neurons significantly smaller than contralateral neurons), and AVCN cell area

abducens nucleus over a 30-min period on a given developmental day. As such, the SH data indicate the level of leucine uptake and incorporation on postnatal day 21 or 23 in animals without a surgically induced hearing loss. For each structure (AVCN, MSO, LSO, and MTB) the level of leucine uptake relative to the abducens nucleus is not significantly different from one another. The same is true for cell area, except that the area of MSO neurons at P21 did differ from P23 animals. 3.2. Protein synthesis and cell area in experimental animals Hearing loss did alter levels of leucine incorporation into central auditory system neurons. Figs. 3 and 4 illustrate within age group comparisons among the experimental conditions. In these figures, abducens corrected protein synthesis values and cell areas are shown for CHL and CA, as are SH values for each auditory structure. The figures are ordered by age/survival and the data arranged first by structure then by bars representing observed values ipsilateral and contralateral to the manipulated ear. P21(21) synthesis: Fig. 3 shows results from P21 animals following a short (6 h) survival time. Within this age group, there were significant differences between CHL and CA manipulations. Ipsilateral AVCN and ipsilateral MSO of CHL animals show significantly higher levels of 1.2

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Fig. 3. Abducens corrected (ABD corr) leucine incorporation values and cell areas for animals measured 6 h after manipulation on P21. * = structures where the ABD corr value was significantly different from sham (P < 0.05). Numerals 1 (ipsilateral AVCN) and 2 (ipsilateral MSO) represent significant differences (P < 0.05) in uptake between CHL and CA conditions.

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K.A. Hutson et al. / Hearing Research 233 (2007) 124–134

eral hearing loss induced at P21. We found significant differences in leucine uptake in AVCN and MTB after 6 h, in MSO after 6 and 48 h. The results of this study demonstrate that hearing loss, whether conductive or sensorineural, alters protein synthesis in central auditory system structures.

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Fig. 4. Uptake values and cell area for CHL and CA animals measured 48 h after manipulation on P21. * = structures where the ABD corr value was significantly different from sham (P < 0.05). Numerals 1 (CHL) and 2 (CA) represent significant differences (P < 0.05) between ipsilateral and contralateral AVCN cells. Numeral 3 represents a significant difference between ipsilateral and contralateral MTB of CHL animals.

was significantly decreased on the ipsilateral side compared to SH animals. In MTB, cells ipsilateral and contralateral to the manipulated ear were significantly smaller than SH animals. Between age group comparisons: Leucine uptake and cell area measures are re-plotted in Fig. 5 to show the pattern of between age group differences in AVCN, MSO, and MTB. Following CHL manipulation on P21, uptake was significantly higher following a 6 h survival than at 48 h survival in both the ipsilateral and contralateral AVCN. There were significant differences in uptake following CHL in MSO bilaterally and in ipsilateral MTB. Following CA, only the ipsilateral MTB demonstrated a significant between age differences in leucine uptake. Between age group variation in the area of auditory brainstem neurons in response CHL and CA were found in MSO ipsilateral and contralateral to the manipulated ear, where P21 animals surviving 6 h were smaller than in animals surviving 48 h. Other between age group differences in cell area were found in ipsilateral AVCN (6 h survival larger than 48 h survival) and contralateral MTB (6 h smaller than 48 h survival). 4. Discussion Cellular incorporation of 3H-leucine was used as a measure of protein synthesis in animals with an abrupt unilat-

The effects of CA or CHL on the cochlear nucleus can be dramatic, yet variable depending upon the species, the developmental state at the time of manipulation, and the type of manipulation performed (see Tucci and Rubel, 2005; Illing et al., 2000). Previous studies using conductive hearing impairment as a model to examine deprivation have described changes that are either similar to CA (e.g., Webster and Webster, 1977; Coleman and O’Connor, 1979; Webster, 1983c; Blatchley et al., 1983), or show virtually no structural changes at all (e.g., Born et al., 1991; Doyle and Webster, 1991; Tucci and Rubel, 1985; Tucci et al., 1987). Our current findings in P21(23) animals support previous reports in demonstrating that CHL, as well as CA, decreases the cell area of ipsilateral AVCN neurons (see Fig. 4). Less is known about the response of superior olivary neurons to hearing loss. In mature animals, CA results in a transneuronal reduction in cell size in the contralateral MTB (Powell and Erulkar, 1962; Jean-Baptiste and Morest, 1975; Pasic et al., 1994) and the ipsilateral LSO (Powell and Erulkar, 1962), while the size of MSO neurons remained qualitatively unchanged. On the other hand, neonatal CHL did not significantly affect cell size in monkey superior olivary nuclei (Doyle and Webster, 1991), yet the size of LSO neurons increased contralateral to CA in neonatal ferrets with no change in size of surviving neurons in the ipsilateral LSO (Moore, 1992). Our results show that the only significant effect of CHL (and CA) on the superior olives was a decrease of cell area in MTB of P21(23) animals (see Fig 4). However, contrary to what would be expected (see Pasic et al., 1994), the MTB ipsilateral to a CHL manipulated ear was significantly smaller than the side contralateral to the manipulation. Our observation of MSO neurons being smaller in P21(21) SH animals than P21(23) SH animals (as well as in the experimental animals, see Fig. 5) suggests that MSO neurons may still be rapidly developing at this time period. In normal rats, MSO neurons expand dramatically in diameter until at least P14, and show a second growth phase between P18 and P30 (Rogowski and Feng, 1981). It could be that at P21 through P23, gerbils are experiencing continued MSO growth. Despite this age difference in MSO cell area seen in our SH animls, our experimental results are similar to Powell and Erulkar (1962), in that we found no evidence for a change in MSO cell area (compared to age matched SH animals) in response to CHL or CA.

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Leucine Uptake AVCN

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Fig. 5. Uptake values and cell area measures grouped by structure to illustrate significant differences between age conditions. For leucine uptake (top graph), numerals 1 = significant difference (P < 0.05) between CHL P21(21) and CHL P21(23) animals in ipsilateral AVCN; 2 = difference between CHL P21(21) and CHL P21(23) animals in contralateral AVCN; etc. Note in AVCN and MSO significant differences are between CHL conditions only. Significant between group differences in MTB (both CHL and CA) are on the side ipsilateral to the manipulation. For cell area (bottom graph), MSO neurons in P21(21) animals are significantly smaller than P21(23) animals regardless of side or manipulation (numerals 2–5). Note other significant differences in cell area are the ipsilateral AVCN (numerals 1) and contralateral MTB (numerals 6) of CHL animals.

4.2. Unilateral hearing loss alters protein synthesis bilaterally in auditory brainstem structures during postnatal development The primary results (Figs. 3 and 4), show that CHL and CA alter the relative level of protein synthesis. This result is virtually the same as previous reports, in that young animals show dramatic central auditory system changes following hearing loss (e.g., see Tucci and Rubel, 2005), independent of the experimental method or species studied. In animals younger than P21, the consequences of hearing loss on protein synthesis may be even more severe (Sie and Rubel, 1992). Furthermore, our results support previous observations that unilateral hearing loss can affect central auditory system structures bilaterally. This has been demonstrated using a variety of methods, including anatomical (e.g., Coleman and O’Connor, 1979), electrophysiological

(e.g., Sumner et al., 2005), metabolic (e.g., Tucci et al., 1999), and neurochemical methods (e.g., Potashner et al., 1997), although there are differences in the severity of the effects of CHL and CA. Within seven days of CA, fiber degeneration is pronounced in the ipsilateral AVCN, and transneuronal fiber degeneration appears in the superior olives and inferior colliculus (Potashner et al., 1997), with no degeneration reported in the contralateral AVCN. After CHL, axonal degeneration only appeared at 112 days post manipulation and was only seen in the AVCN, but interestingly, bilaterally in AVCN (Potashner et al., 1997). This raises the possibility that CHL ultimately has a greater effect on the contralateral AVCN than does CA. After CA, there is a rapid loss of inputs on the side of the ablation, followed by an up-regulation of GAP-43 (Illing et al., 1997; Kraus and Illing, 2004) and the emergence of new synaptic

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contacts on neurons in the AVCN. However, CHL does not does not prompt synaptogenesis in the deafferented cochlear nucleus (Benson et al., 1997). Over time, CHL and CA reduce protein synthesis in auditory structures: The earliest response to unilateral hearing loss that we measured (6 h) suggests that CHL promotes a transient increase in protein synthesis in MSO and MTB. Despite this early change in P21 animals 6 h after hearing loss, there is a progression toward a significant decrease in protein synthesis by 48 h. Thus there appears to be an initial response to deafferentation at 6 h which may represent an early attempt by central auditory structures to re-set or re-balance to a sudden change in inputs, followed by a decline towards decreased synthesis 48 h post hearing loss. Similar observations have been reported in the visual system of young monkeys (Kupfer and Downer, 1967). Subsequent to unilateral denervation, neurons in the affected lateral geniculate nucleus show increased leucine incorporation over the first 24 h, then decrease incorporation over the following 17–21 days. In our material, significant changes in the central auditory system response to hearing loss began sooner in the superior olives than in the cochlear nucleus. Our observations that MSO and MTB show bilateral changes in response to CHL or CA are not unprecedented, and similar results have been seen using 2-DG (e.g., Tucci et al., 1999) and neurochemical methods (e.g., Potashner et al., 1997; Suneja et al., 1998a). Leucine metabolism post-unilateral hearing loss: Though our results show a significant decline in protein synthesis in P21 animals after 48 h, auditory neurons never ceased to incorporate leucine. We have no means to determine the destiny of the newly-generated proteins. Changes in leucine uptake over time would measure, among other things, an altered demand for membrane construction and maintenance. For example, in MSO, there are changes in dendritic morphology and dendritic atrophy following hearing loss (e.g., Feng and Rogowski, 1980; Russell and Moore, 1999; Tucci et al., 2001). A decreased metabolic demand would also result from the pruning of axon arbors due to transneuronal degeneration (e.g., Kim et al., 1997; Morest and Bohne, 1997; Potashner et al., 1997; Russell and Moore, 2002), and transneuronal cellular atrophy (JeanBaptiste and Morest, 1975; Hashisaki and Rubel, 1989; Pasic et al., 1994; Lesperance et al., 1995; Tierney et al., 1997). However, a portion of the leucine uptake is likely used in metabolic pathways related to up- and down-regulation of neurotransmitters and their receptors, which undergo significant bilateral changes after hearing loss (e.g., Potashner et al., 1997, 2000; Suneja et al., 1998a,b, 2000), as well as the expression of proteins, such as calcium binding proteins (e.g., Caicedo et al., 1997; Fo¨rster and Illing, 2000), GAP-43 (Michler and Illing, 2002; Kraus and Illing, 2004) and synaptophysin (e.g., Benson et al., 1997) which are also up- and down-regulated following hearing loss. Jin and Godfrey (2006) found that CA affects muscarinic acetylcholine receptor binding in the AVCN

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bilaterally, where binding increases steadily from 7 days to 2 months post ablation, with a 42% increase in the contralateral AVCN. This suggests the possibility of significant modulation of activity within neurons of AVCN via intracellular G-proteins. The existing evidence (summarized above) indicates that CHL has a substantial effect on central auditory system structures, including altered neurotransmitter metabolism and possible axonal changes. Furthermore, some changes were bilateral, and included the MSO, just as in our leucine uptake material. The bilateral nature of these observed effects, suggests that unilateral conductive hearing impairment must affect processes related to binaural sound processing, and ultimately affect higher order central auditory system structures (Clements and Kelly, 1978; Potash and Kelly, 1980; Kelly and Potash, 1986; Tucci et al., 1999). 4.3. Binaural consequences of unilateral conductive hearing loss In behavioral investigations on the effects of unilateral CHL in young animals, a reduction in hearing by as little as 10 dB results in significant sound localization deficits (Potash and Kelly, 1980). However, animals with bilateral CHL do not show localization deficits. This has been demonstrated in rats (Potash and Kelly, 1980), guinea pigs (Clements and Kelly, 1978), and gerbils (Kelly and Potash, 1986). Thus, a binaural imbalance induced by unilateral CHL appears to have unique consequences on the central auditory system as compared to bilateral CHL. Furthermore, behavioral deficits in sound localization persist after alleviation of unilateral CHL in young animals (e.g., Clements and Kelly, 1978; Knudsen et al., 1984) and after repair of congenital unilateral CHL in humans (e.g., Wilmington et al., 1994). An important finding here is that MSO and MTB are the major sites of early changes in protein synthesis subsequent to CHL. Both MSO and MTB are important structures involved in sound localization, and for constructing a central auditory system representation of auditory space (e.g., Jenkins and Masterton, 1982; Glendenning and Masterton, 1983; Glendenning et al., 1992). Furthermore, unilateral deprivation induces rapid up- and down-regulation in the expression of calcium binding proteins in both MSO and MTB (Caicedo et al., 1996). Our observation that MSO ipsilateral to the P21(21) CHL manipulated ear showed the greatest increase in uptake of any auditory brainstem structure (followed by a rapid decline in uptake after 48 h) suggests that MSO may be exceptionally susceptible to the effects of unilateral CHL. Studies on the expression of calcium binding proteins in the central auditory system, calretinin in particular, provide some supportive evidence for this conclusion. Calretinin is found in central auditory system structures related to pathways important for sound localization (e.g., Caicedo et al., 1996), and calretinin immunoreactivity is high in the neuropil and neu-

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rons of AVCN, LSO, and MSO (Caicedo et al., 1996; Alvarado et al., 2004; Fuentes-Santamaria et al., 2005). Caicedo et al. (1997) demonstrated that within 6 h of temporary deafferentation (by intracochlear AMPA perfusion), there was a significant reduction in calretinin staining only in neurons of the ipsilateral MSO. This observation, together with our leucine uptake findings, imply that activity in MSO is indeed affected immediately (within 6 h) after a unilateral CHL. MSO receives excitatory inputs, in similar amounts, from each cochlear nucleus (e.g., Glendenning et al., 1985) and participates in the analysis of timing cues relevant for sound localization (Yin and Chan, 1990). Given the projection to MSO from AVCN, CHL should have reduced activity in both MSO’s by roughly equal amounts, and at longer survival times this is what is seen in 2-DG data (Tucci et al., 1999). Yin and Chan (1990) found that MSO neurons not only are sensitive to interaural time differences, but also that MSO neurons preferred (i.e., had maximum firing rates) when the time differences correspond to a sound stimulus located in the contralateral sensory hemifield. Consequently, MSO projections to the inferior colliculus carry information about the location of a sound source on the opposite side. Thus unilateral CHL may alter the activity in MSO neurons, which could enhance the representation of the contralateral auditory field at higher central auditory system levels. Errors in sound localization made by gerbils with one ear blocked imply the same interpretation – that the auditory world has been altered to favor the contralateral hemifield (Kelly and Potash, 1986). Alternatively, all or a portion of the up-regulation of protein synthesis in MSO might be due to increased structural demands. In measurements of cytochrome oxidase activity in young animals subsequent to unilateral CHL, Tucci et al. (2001) found evidence for changes in the length of MSO dendrites. Surprisingly, CHL increased dendritic length, while unilateral CA decreased dendritic length. Up-regulation of protein synthesis in MSO at 6 h after CHL may then be attributable to neurons preparing for dendritic expansion. Nonetheless, the primary conclusion remains that MSO neurons show a rapid and significant response to unilateral CHL. While unilateral CA eliminates all inputs to the central auditory system from one ear, unilateral CHL only diminishes the inputs from one ear. Thus, the residual function in the CHL ear may evoke compensatory adjustments by the contralateral cochlear nucleus (Sumner et al., 2005). Acknowledgements The authors would like to thank Jason Maloney and Adam Graff for their technical contributions during the tissue analysis phase of this report. Grant Sponsor: National Institute of Health/National Institute on Deafness and Other Communication Disorders; Grant Number: DC05416 to DLT.

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