Effects of cochlear ablation on amino acid concentrations in the chinchilla posteroventral cochlear nucleus, as compared to rat

Neuroscience 154 (2008) 304 –314

EFFECTS OF COCHLEAR ABLATION ON AMINO ACID CONCENTRATIONS IN THE CHINCHILLA POSTEROVENTRAL COCHLEAR NUCLEUS, AS COMPARED TO RAT D. A. GODFREY,* K. CHEN,1 M. A. GODFREY, Y.-M. JIN,1 K. T. ROBINSON AND C. HAIR

It is well established that damage to peripheral sensory organs can lead to changes in the CNS, including chemical changes related to neurotransmission from the primary sensory nerve to the first central nucleus of the system (Wall et al., 2002). The auditory nerve carries information about sounds from the cochlea of the inner ear to the cochlear nucleus in the brain. The innervation of the cochlear nucleus by the auditory nerve has been studied in several mammals, including cats, rats, guinea pigs, and chinchillas (Harrison and Irving, 1966; Osen, 1970; Noda and Pirsig, 1974; Lorente de Nó, 1981; Morest et al., 1997; Ryugo, 2008). Effects of removal of this auditory nerve innervation on chemistry in the cochlear nucleus have been reported mostly for guinea pigs (Fisher and Davies, 1976; Wenthold and Gulley, 1977; Wenthold, 1978; Potashner et al., 1985, 1997, 2000; Suneja et al., 2000), including effects on concentrations of the probable auditory nerve neurotransmitter, glutamate (Wenthold and Gulley, 1977; Wenthold, 1978). In our studies of effects of the ototoxic drug carboplatin on amino acid concentrations in the cochlear nucleus of chinchillas (Li et al., 2002; Godfrey et al., 2005), we found effects to be more slowly developing than those of cochlear ablation in guinea pigs (Wenthold, 1978) or rats (Godfrey et al., 2004a). Because the studies were done on different species of mammals, however, we could not determine how much of the differences in timing were related to the different treatments and how much to the different species. We have therefore carried out more complete measurements of the effects of cochlear ablation on amino acid chemistry in the cochlear nucleus of chinchillas for comparison to the effects of carboplatin. Further, to enable a more detailed analysis, we have focused on just the posteroventral subdivision of the cochlear nucleus (PVCN). The effects of carboplatin on glutamate and aspartate concentrations were largest in the PVCN and showed the best correlation with extent of inner hair cell loss (Li et al., 2002; Godfrey et al., 2005). This may relate to its dense innervation from the auditory nerve, especially in its caudal portion (Osen, 1970; Lorente de Nó, 1981; Morest et al., 1997), which contains a rather homogeneous population of the large somata and extensive dendritic trees of one type of neuron (Harrison and Irving, 1966; Osen, 1969; Morest et al., 1990), known as octopus cells (Osen, 1969). Octopus cells have been well studied in terms of their structure (Osen, 1969; Kane, 1973; Smith et al., 2005), extensive innervation from the auditory nerve (Osen, 1970; Morest et al., 1973; Kane, 1973, 1974, 1979),

Division of Otolaryngology and Dentistry, Department of Surgery, Mail Stop 1092, University of Toledo Health Science Campus, 3000 Arlington Avenue, Toledo, OH 43614, USA

Abstract—Using a microchemical approach, we measured changes of amino acid concentrations in the chinchilla caudal posteroventral cochlear nucleus (PVCN) after cochlear ablation to determine to what extent slow decreases of glutamate and aspartate concentrations after carboplatin treatment resulted from slower effects of cochlear damage in chinchillas than in rats and guinea pigs, as opposed to effects of carboplatin treatment being slower than those of cochlear ablation. Our results indicate that both factors are involved: decreases of glutamate and aspartate concentrations after cochlear ablation are much slower in chinchillas than in rats and guinea pigs, but they are much faster than the decreases after carboplatin treatment. Further, aspartate and glutamate concentrations in the chinchilla caudal PVCN decreased by larger amounts after cochlear ablation than in rats or guinea pigs, and there was a transient increase of aspartate concentration at short survival times. Detailed mapping of amino acid concentrations in the PVCN of a chinchilla with 1 month survival after cochlear ablation and a rat with 7 days’ survival indicated that the reductions of glutamate and aspartate occurred throughout the PVCN but were somewhat larger in ventral and caudal parts in chinchilla. Any decreases in the adjacent granular region were very small. There were also sustained bilateral decreases in concentrations of other amino acids, notably GABA and glycine, in the caudal PVCN of cochlea-ablated chinchillas but not rats. The effects of cochlear ablation on the concentrations of most of these other amino acids in chinchilla caudal PVCN differed from those of carboplatin treatment. Thus, although a major effect of auditory nerve damage on the cochlear nucleus— decreases of glutamate and aspartate concentrations— occurs across species and types of lesions, the details of timing and magnitude and the effects on other amino acids can vary greatly. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: aspartate, auditory, GABA, glycine, glutamate, taurine.

1

Present address: K. Chen, Hough Ear Institute, Oklahoma City, OK, USA; Y.-M. Jin, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. *Corresponding author. Tel: ⫹1-419-383-3571; fax: ⫹1-419-383-3096. E-mail address: [email protected] (D. A. Godfrey). Abbreviations: ChAT, choline acetyltransferase; HPLC, high-performance liquid chromatography; PVCN, posteroventral cochlear nucleus; VCN, ventral cochlear nucleus.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.12.031

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electrophysiology (Godfrey et al., 1975; Golding et al., 1995; Smith et al., 2005), and axonal projections (Warr, 1969; Adams, 1997; Smith et al., 2005). We have used microdissection combined with highperformance liquid chromatography (HPLC) to map concentrations of amino acids, including the neurotransmitters glutamate, glycine, and GABA, in the caudal PVCN of chinchillas killed at different survival times after cochlear ablation and compared results with those in rats. Further, we have mapped three-dimensionally the effects in both PVCNs of one chinchilla and one rat to examine the changes at different locations within the PVCN and adjacent granular region.

EXPERIMENTAL PROCEDURES Most of the procedures used in this study have been described previously (Godfrey and Matschinsky, 1976; Ross et al., 1995; Godfrey et al., 2000, 2005).

Animals and cochlear ablations Chinchillas were obtained from Jarr Chinchilla, Hubbard, OH, USA. Seventeen male chinchillas weighing 496 – 647 g were divided into several groups. Eleven received surgery for unilateral (right) cochlear ablation; two of these were killed 3 days later, three 7 days later, one 15 days later, two 1 month (31 days) later, and three 3 months (84 days) later. Two received surgery for bilateral cochlear ablation and were killed 14 and 30 days later. Four received sham surgery and were killed 3, 7, 31, and 83 days later. The relatively small numbers of animals used to cover the several survival times relate to the tedious nature of these experiments, our previous experience indicating that the trends in amino acid concentrations are very consistent among individual animals of the same species, combined with the capability of the microchemical approach to obtain many samples for each animal, and our goal to minimize animal use. Under halothane-plus-oxygen inhalation anesthesia (halothane, USP, from Halocarbon Laboratories, River Edge, NJ, USA), using aseptic procedures, a post-auricular incision was made to expose and open the bulla, then the cochlea was destroyed by drilling through it. Buprenorphine (0.01– 0.02 mg/kg) (from Reckitt Benckiser Healthcare (UK), Ltd.) was given s.c. just before and 12 h after surgery. Oxytetracycline (20 mg/kg) (from Pfizer Animal Health) was given intramuscularly immediately after surgery and for 2 days afterward. The chinchillas with bilateral surgery recovered more slowly than those with unilateral surgery and showed no startle responses to sound. They were given 20 ml of dextrose s.c. 2 days after surgery, 35 ml on the next day, and 20 ml on the fourth postoperative day, as well as 10 ml of normal saline intraperitoneally on the third and fourth postoperative days, after which they recovered well. The sham surgery was the same as the ablation surgery through opening the bulla, but no drilling was done to destroy the cochlea. The postoperative treatment was the same as for the chinchillas with unilateral cochlear ablation surgery. The rat tissues used for this study had been prepared for previous studies of cochlear ablation effects on the cholinergic system (Jin et al., 2005; Jin and Godfrey, 2006). Male Sprague– Dawley rats (275–299 g, 60 days of age) were obtained from Harlan Sprague Dawley, Indianapolis, IN, USA. Control rats received no surgery. In the lesioned rats, the left cochlea was ablated as described previously (Jin et al., 2005; Jin and Godfrey, 2006), and rats were killed at various times afterward. Samples of PVCN were obtained from both sides of two control rats (one age-matched to the 7-day-survival rats and one to the 2-month-survival rats), one rat with 2 days’ survival, and three rats each with 7 days’, 1 month’s (31–33 days), and 2 months’ (58 – 62 days) survival.

Fig. 1. Photographs of right temporal bone (top) and a Thionin-eosinstained section through the right cochlea of chinchilla K, which survived 31 days after right cochlear ablation surgery, and of chinchilla R, which survived 83 days after sham surgery on the right side. The protrusion of the cochlea (C) can be seen in the temporal bone of chinchilla R but not that of chinchilla K. Parts of the spiral ganglion (G) and auditory nerve (N) can be seen in the section of chinchilla R but not that of chinchilla K. Extensive Thionin-stained debris and blood fill the cochlear scalae of chinchilla K. At the bottom, the scale bar⫽1 mm in the sections.

Evaluation of lesions For the rats, the cochlear ablations were evaluated by visual inspection of the temporal bones after animals were killed. For the chinchillas, the cochlear ablations were evaluated by visual inspection of the temporal bones after animals were killed and by observations of Nissl-stained sections cut from the decalcified and paraffin-embedded temporal bones (Fig. 1). On this basis, all but two cochlear ablations were judged to be complete. One ablation judged to be incomplete was the first one attempted, and this animal was therefore not included in the study. The other ablation judged to be incomplete was for the left side of the chinchilla with bilateral surgery and 30 days’ survival.

Isolation of tissue samples Rats and chinchillas were killed by decapitation while deeply anesthetized with sodium pentobarbital (50 – 80 mg/kg) (Abbott, Chicago, IL, USA), then their brains (entire for rats and hindbrain portion for chinchillas) were isolated and frozen within 3–5 min of death in Freon (Fisher Friendly Freeze-It, Fisher Scientific, Hampton, NH, USA) chilled to its freezing point with liquid nitrogen. Frozen tissue blocks were stored at ⫺80 °C in double airtight containers until sectioning. Transverse sections of frozen brains were cut 20 ␮m thick at ⫺20 °C in a cryostat. For chinchillas, every fourth section was placed into aluminum racks kept at ⫺80 °C until they were subsequently put inside glass vacuum tubes for freeze drying (Lowry

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and Passonneau, 1972). The three other groups of every fourth section were mounted onto glass slides for staining with Cresyl Violet for Nissl substance, for acetylcholinesterase activity, and for cytochrome oxidase activity (Godfrey and Matschinsky, 1976; Godfrey et al., 2005). For rats, every third section was saved for freeze drying and the other two groups of every third mounted on slides and stained for Nissl substance and either acetylcholinesterase or cytochrome oxidase activity. For the rats, sections near the center of the rostrocaudal extent of the PVCN were mounted onto slides for a receptor binding study (Jin and Godfrey, 2006). Freeze-dried sections were stored in the glass tubes under vacuum below ⫺20 °C. Dissection of freeze-dried tissue into samples for assay was done at 25⫻ magnification, in a room with relative humidity maintained at or below 50%. A drawing attachment on the Wild dissecting microscope was used to map sample locations (Godfrey and Matschinsky, 1976). Samples were weighed on quartz-fiber microbalances (Lowry and Passonneau, 1972) and then loaded into 300 ␮l-capacity glass tube inserts (from Waters Corporation, Milford, MA, USA) for HPLC measurement of amino acids. Samples from both PVCNs of the same animal were included in the same assay, and control animals were interspersed with lesioned ones to guard against any differences resulting from changes in assay results over time.

Amino acid assay For HPLC assay of free amino acid concentrations, 50% (vol/vol) methanol was added to the samples to extract the amino acids, then aliquots were withdrawn by a WISP autosampler and derivatized with ortho-phthaldialdehyde (Fluoraldehyde Reagent from Pierce Chemical Company, Rockford, IL, USA). The fluorescent derivatized amino acids were separated by reversed-phase chromatography on a C8 column (from Mac-Mod Analytical, Chadds Ford, PA, USA), using gradient elution with a Waters HPLC system. The eluate was passed through a Spectrovision fluorescence detector (Groton Technologies, Boxborough, MA, USA), and the peaks of fluorescence were quantitated via a computer using Millennium software (from Waters). Amino acids were identified by their retention times, and their concentrations were calculated by comparison to calibrated amino acid standard solutions included in the same assays (Hill et al., 1979; Ross et al., 1995; Godfrey et al., 2000, 2005). Standards were prepared from Standard H (from Pierce) supplemented with calibrated amounts of asparagine, glutamine, taurine, and GABA (from Sigma, St. Louis, MO, USA). Other reagents were from Sigma or from Fisher Scientific. Although measurements were made for 12 amino acids, data for asparagine, alanine, and tyrosine are not reported because they were not sufficiently reliable in the assays for this study.

PVCN volume and tissue density Volumes of cochlear nucleus regions on both left and right sides were measured in all chinchillas by tracing the boundaries in stained sections, mostly those with the Nissl stain, scanning the tracings into a computer, and tracing over the boundaries, using Neurolucida software (obtained from MicroBrightfield, Colchester, VT, USA), so that the area of each cochlear nucleus region could be measured in each section. Total volumes were calculated by multiplying the areas times the distances between sections (difference in section number times 20 ␮m). Data for PVCN and total ventral cochlear nucleus (VCN) are presented here. Volumes could not be measured for rat PVCN because of the missing blocks of sections. During degeneration of a fiber tract after its transection, there is breakdown and removal of myelin from the tissue. Our previous study suggested that this began within a month in the vestibular nerve root after vestibular ganglionectomy (Godfrey et al., 2004b). The density of freeze-dried brain tissue, as dry weight per volume,

is strongly related to lipid content, particularly the lipid in myelin (Godfrey and Matschinsky, 1976). Since the concentrations of the amino acids are expressed per dry weight and since they are predominantly associated with the non-lipid portions of tissue, decreases in tissue density at later times after the transection of the auditory nerve during cochlear ablation could lead to apparent increases in amino acid concentrations. Measurements of PVCN tissue density at the different times after cochlear ablation were therefore made in the dissected sections to enable corrections for such apparent increases. Tissue density, as dry weight per volume, was determined by dividing the total dry weight of PVCN in each section (sum of sample weights) by the total volume of PVCN dissected (total area of the dissected portion times the 20 ␮m section thickness). In a few cases, the lipid weight of tissue samples was directly measured in additional sections by extracting the lipid from weighed samples with ethanol and hexanes, then reweighing the samples (Lowry and Passonneau, 1972; Godfrey and Matschinsky, 1976).

Data presentation Concentrations of amino acids, as mmol/kg dry weight, were plotted onto the maps of the dissected sections. Also, the data for all samples within the caudal PVCN, or other region, were averaged for each group. Differences from control values were evaluated for statistical significance by analysis of variance and t-tests. Data for both sides of control rats and sham-lesioned chinchillas were combined for these comparisons since there were no statistically significant differences between them. Based on the Bonferroni correction (Pagano and Gauvreau, 1993), the significance level for considering a difference statistically significant in t-tests was made more stringent according to the number of comparisons to the control or sham value.

Treatment of animals Treatment of animals was approved by the University of Toledo Health Science Campus Institutional Animal Care and Use Committee and in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize animal suffering as well as the number of animals used.

RESULTS Effects of cochlear ablation on PVCN structural parameters The volume of the PVCN on the lesioned side appeared to decrease by 84 days after unilateral cochlear ablation (Table 1). Because of the small numbers of measurements (one per cochlear nucleus) and sizable variation in PVCN volume even among sham animals, analysis of variance indicated no significant differences among the groups, even though the average at 84 days was significantly different from the sham average by t-test (P⫽0.002). The consistency of the volume reduction at 84 days for the total VCN as well as the PVCN suggests that the difference from sham is real. The density of the chinchilla PVCN tissue on the lesioned side (expressed as dry weight per volume) increased somewhat relative to the sham average at 31 days after unilateral cochlear ablation and decreased at 84 days (Table 1). The decrease at 84 days was entirely accounted for by loss of lipid weight, since the non-lipid dry weight per volume was the same as the sham average.

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Table 1. Volumes of chinchilla PVCN and densities of caudal PVCN at different times after right cochlear ablation Survival and surgery

Side

PVCN volume (␮l)

Total VCN volume (␮l)

Dry wt/volume (g/l)

Lipid-free dry wt/vol (g/l)

Sham 3 Days right

R⫹L R L R L R L R L R L R L R L

0.86⫾0.07 (8) 0.71 (2) 1.00 (2) 0.97⫾0.25 (3) 0.99⫾0.11 (3) 0.70 (1) 0.65 (1) 0.83 (1) 0.96 (1) 0.84 (1) 0.77 (1) 0.70 (2) 0.90 (2) 0.51⫾0.03 (3) 0.90⫾0.14 (3)

2.50⫾0.13 (8) 2.63 (2) 2.39 (2) 2.76⫾0.16 (3) 2.72⫾0.13 (3) 2.04 (1) 2.00 (1) 2.12 (1) 2.79 (1) 1.96 (1) 2.23 (1) 2.15 (2) 2.96 (2) 1.63⫾0.18 (3) 2.60⫾0.21 (3)

379⫾5 (16) 439 (2) 416 (2) 411⫾26 (4) 398⫾14 (4) 425 (1) 410 (1) 416 (2) 418 (2) 368 (2) 422 (2) 433⫾13 (8)* 396⫾13 (8) 320⫾10 (6)** 409⫾19 (6)

165⫾2 (6)

7 Days right 14 Days bilateral 15 Days right 30 Days bilateral 31 Days right 84 Days right

150⫾3 (3) 147⫾1 (3) 161⫾7 (3) 163⫾1 (3)

Data are means⫾S.E.M. (number of measurements), involving all chinchillas except for lipid-free dry wt/vol. Lipid-free dry wt/volume data are from one sham, one 31-day, and one 84-day survival lesioned chinchilla. Values for both sides of sham-lesioned animals were grouped since there were no statistically significant differences between them. Statistical comparisons were not made among lipid-free dry wt/vol data. * P⬍0.005, statistical significance for comparison with sham. ** P⬍0.001, statistical significance for comparison with sham.

The density of the rat PVCN tissue could not be measured with confidence because of some uncertainty about section thickness, but the ratios of densities for lesionedand control-side PVCN in the same section were calculated: 1.13⫾0.025, 0.94⫾0.076, and 0.92⫾0.090 for three measurements each at 7, 32, and 60 days after unilateral cochlear ablation, respectively. There were no significant differences among the groups. Effects of cochlear ablation on caudal PVCN excitatory neurotransmitter amino acid concentrations The concentrations of glutamate and aspartate in the lesioned-side caudal PVCN of rats decreased significantly compared with those in control rats, by 37% and 26%, respectively, by 2 days after unilateral cochlear ablation, and remained at values close to 35% reduction through 32 days, followed by some recovery at 60 days (Fig. 2). The increased concentrations at 60 days, as compared with 32 days, may relate to some decrease of PVCN size at that time (Jin and Godfrey, 2006) but apparently not to changes in tissue density. They occurred for all the amino acids except serine and arginine (Fig. 2). There were no significant changes of glutamate or aspartate concentration in the contralateral PVCN, although there was a trend for decreases over time. In the chinchillas, at 15– 84 days after unilateral cochlear ablation, glutamate and aspartate concentrations were decreased in the lesioned-side caudal PVCN, by even larger amounts than in rats: by about two-thirds at 31 days’ survival (Fig. 2). Roughly 50% increases of glutamate and aspartate concentrations in the lesioned-side PVCN at 84 days’ survival, as compared with 31 days’ survival, correlated partially with the decrease in tissue density between these times (Table 1). There were possibly some small reductions of aspartate and glutamate concentrations in the contralateral caudal PVCN, but the

only statistically significant reduction was for glutamate at 31 days’ survival. For shorter times after unilateral cochlear ablation, the results for rats and chinchillas showed some noticeable differences. The decrease of glutamate concentration in the lesioned-side caudal PVCN developed much more gradually in chinchillas than in rats, not approaching its maximal amount until 15 days after surgery, a time seven times longer than for rats. For aspartate, there were even more differences. Surprisingly, the average aspartate concentration increased in the lesioned-side caudal PVCN, by more than 50%, at both 3 and 7 days after surgery, before it decreased to about half the sham value at 15 days. This transient increase occurred in all five chinchillas having these shorter survival times. The data for both sides of the chinchilla with bilateral cochlear ablation and 14 days’ survival (Fig. 2) were generally similar to those for the lesioned-side caudal PVCN of the chinchilla with 15 days’ survival after unilateral cochlear ablation. For the chinchilla with 30 days’ survival after bilateral cochlear ablation surgery, which failed to completely ablate the left cochlea, the results for glutamate and aspartate in the right PVCN did not differ greatly from those for the chinchillas with unilateral cochlear ablation and 31 days’ survival. The concentration of glutamine, a major substrate for synthesis of glutamate (Cooper et al., 1996), changed much less than that of glutamate after cochlear ablation in rat or chinchilla and showed no preferential decrease on the lesioned side (Fig. 2). Effects of cochlear ablation on caudal PVCN inhibitory neurotransmitter and other amino acid concentrations Both glycine and GABA concentrations decreased bilaterally in the chinchilla caudal PVCN after unilateral cochlear

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Fig. 2. Amino acid concentrations, as ratios to sham-lesioned Chin or control rat average, are plotted vs. time after cochlear ablation surgery for both Les and Contr caudal PVCN of Chin and rats, identified by symbol shape and color according to the legend shown at lower right in the arginine graph (Chin, chinchilla; Contr, contralateral; Les, lesioned side). Data for individual sides of sham-lesioned Chin and control rats are plotted at the zero time in each graph. Sham-lesioned Chin and control rat amino acid concentrations, in mmol/kg dry wt, are (both sides combined in each case), as mean⫾S.E.M., for 56 –57 samples in Chins and 16 samples in rats, respectively, for aspartate 17.3⫾0.7 and 13.1⫾0.7, glutamate 32.9⫾0.7 and 27.8⫾1.0, glutamine 22.0⫾0.5 and 19.3⫾0.9, glycine 9.6⫾0.3 and 16.8⫾0.8, GABA 3.0⫾0.2 and 4.3⫾0.3, taurine 11.3⫾0.2 and 10.4⫾0.5, serine 3.9⫾0.1 and 4.7⫾0.4, threonine 3.9⫾0.1 and 6.5⫾0.4, arginine 1.0⫾0.05 and 2.3⫾0.1. Numbers of samples for lesioned-side (right) and Contr (left) PVCN of Chins at each survival time are, respectively, 10 and 10 for two Chins at 3 days, 24 and 18 for three Chins at 7 days, 14 and 13 for one Chin at 15 days, 13 and 16 for two Chins at 31 days, and 18 and 19 for three Chins at 84 days. Numbers of samples for lesioned-side (left) and Contr (right) PVCN of rats at each survival time are, respectively, 4 and 10 for one rat at 2 days, 14 and 18 –19 for three rats at 7 days, 14 –15 and 21 for three rats at 32 days, and 10 and 16 –17 for three rats at 60 days. Two data points are included for Chins at 84 days after surgery: one corrected, based on the data in Table 1, for the difference in dry weight per volume as compared with Contr (the lower point, which is connected by the line), and one uncorrected. Extra data points for Chins at 14 days and at 30 days are for both sides of one Chin at each survival time having bilateral cochlear ablation surgery. The symbol for the right PVCN in each case is colored as for the right (lesioned-side) and the left as for the left (Contr) PVCN of the Chins with unilateral cochlear ablations. Statistically significant differences from sham (Chin) or control (rat) averages are indicated by letters, of the same color as the symbol to which each pertains (the one it is closest to): a for P⬍0.005, b for P⬍0.001, and c for P⬍0.0001.

ablation, whereas changes were much less in rat (Fig. 2). The decreases in chinchilla began at 3 days and reached more than 40% at 31 days’ survival. The concentration of serine, a substrate for glycine synthesis (Cooper et al., 1996), showed some evidence of bilateral, especially contralateral, decreases in the chinchilla caudal PVCN after cochlear ablation, but not in rats, for which there were trends toward increases (Fig. 2). Concentrations of taurine, threonine, and arginine decreased bilaterally in the chinchilla caudal PVCN after cochlear ablation (Fig. 2), whereas there were few signif-

icant changes in rats. Taurine concentrations showed the steepest lesioned-side increases at the longest survival time in both rat and chinchilla (Fig. 2). Except for serine and arginine, the results for the chinchilla with 14 days’ survival after bilateral cochlear ablation were similar to the lesioned-side results for the chinchilla with 15 days’ survival after unilateral ablation (Fig. 2). However, for the chinchilla with 30 days’ survival after bilateral surgery, there were significant increases in glycine, taurine, serine, and threonine concentrations in the right PVCN, the side with the successful surgery (Fig. 2).

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Fig. 3. Distributions of glutamate concentrations in the PVCNs of a rat that was killed 7 days after surgery to ablate the left cochlea. Both PVCNs, from the same section in each case, were dissected in similar ways to enable paired comparisons between samples on the left and right sides. For the right (control-side) PVCN, the measured glutamate concentrations are mapped using the red– orange–yellow color code. For the left (lesionedside) PVCN, glutamate concentrations are mapped as percentages of those in corresponding samples on the right, using the blue– green–yellow color code. The asterisk marks a sample of the left PVCN for which data were not obtained. Higher-numbered sections are more rostrally located, and the distances between sections can be calculated as 20 ␮m times the difference in section number. Sections between numbers 24 and 39 were used for receptor binding experiments (Jin and Godfrey, 2006). Thin lines are cut edges of samples. Thick lines are regional boundaries, including solid lines for outside borders, dashed lines for histological boundaries observed within the dissected freeze-dried sections, and dotted lines for histological boundaries traced from adjacent Nissl-stained sections. Regional abbreviations are: G, granular region; I, interstitial nucleus (auditory nerve root); P, PVCN. Directional abbreviations, on the 1 mm scale, are D, dorsal; L, lateral; M, medial; V, ventral.

Effects of unilateral cochlear ablation on detailed distributions of amino acid concentrations in the PVCN To examine the effects of cochlear ablation on amino acid concentrations in the entire PVCN and adjacent granular region at higher resolution, more detailed mapping was done for one rat and one chinchilla having survival times at which the changes were well established: 7 days for the rat and 31 days for the chinchilla. In the rat, glutamate concentrations in the control-side (right) PVCN were generally somewhat higher rostrally than caudally and ventrally (Fig. 3). Concentrations in the granular region were higher than in the PVCN, and concentrations in the interstitial nucleus were lower. On the lesioned side, there was little if any reduction of glutamate concentration in the granular region. The largest reductions occurred in the interstitial nucleus. Within the PVCN, there were no clear differences among subregions in the amount of reduction of glutamate concentration, except for some smaller reductions in the most caudal section. In the chinchilla, glutamate concentrations in the control-side (left) PVCN (Fig. 4) had a similar range as those in the rat (Fig. 3). As in the rat, they were generally higher rostrally than caudally or ventrally and lowest in the interstitial nucleus (Fig. 4). However, concentrations in the granular region were generally higher than in the rat. On the lesioned side, there was little if any reduction of glutamate concentration in the granular region but a large reduction in the interstitial nucleus. Within the PVCN, there was a clear trend for larger percentage reductions ventrally and caudally (see Discussion). In order to obtain average amino acid concentrations for chinchilla PVCN and granular subregions, data for most

of the mapped samples on both sides were grouped into granular and PVCN subregions as defined in an atlas of the chinchilla cochlear nucleus (Morest et al., 1990), by comparison of the dissected section locations and the appearances of the adjacent Nissl-stained sections with those of the atlas. For glutamate and aspartate, for which there were clear differences between lesioned and control sides, these were evaluated (Fig. 5). For the other amino acids, where there were not clear differences between the two sides, only control-side data were evaluated (Fig. 6). Aspartate was the only amino acid for which control-side (left) concentrations in granular subregions were lower than for most PVCN subregions (Fig. 5). Besides the obvious differences between granular and PVCN subregions, the lesioned-side/contralateral ratio for both aspartate and glutamate was significantly higher in the anterior part than in the central part (octopus cell region) or ventral part (P⬍0.001 for aspartate and P⬍0.0001 for glutamate) of the PVCN, whereas the differences in magnitude between contralateral and lesioned-side concentrations showed no significant differences among PVCN subregions. The average control-side concentrations of GABA and glycine for the PVCN ventral part were significantly lower than those for the central part (Fig. 6), and, for glycine, also the anterior part (P⫽0.0001).

DISCUSSION Considerations in interpreting the results Although the most prominent changes in amino acid concentrations should be closely associated with the degeneration of auditory nerve fibers, the spatial resolution of the

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Fig. 4. Distributions of glutamate concentrations in the PVCNs of chinchilla K, which was killed 31 days after ablation of the right cochlea. Both PVCNs, from the same section in each case, were dissected in similar ways to enable paired comparisons between samples on the left and right sides. The regional boundary within the granular region separates the more lateral plexiform layer from the granule cell layer (Morest et al., 1990). Color codes and other details are as in Fig. 3.

microchemical approach as applied here does not enable determination of which other neurons or glia may also be the sites of changes. However, previous measurements have suggested that concentrations of aspartate, glutamate, and serine are at least twice as high in neurons and GABA more than 10 times as high; glutamine and taurine twice or more as high in glia; and threonine and arginine similar in neurons and glia (Patel and Hunt, 1985; Ottersen et al., 1992; Hassel et al., 1995). The higher glycine concentrations reported for glia than for neurons (Patel and Hunt, 1985) are probably not relevant to the cochlear nucleus because they were measured for cultures of the cerebellum, which has low glycine concentrations (Ross et al., 1995). Thus, concentration changes of GABA are likely to be totally related and aspartate, glutamate, and serine mostly related to neurons, whereas changes in glutamine and taurine concentrations may be mostly related to glia. For example, the increases of glutamine and taurine concentrations at the longest survival times may be related to proliferation of astrocytic processes at these times (Kane, 1974). Across-species comparisons of the effects of cochlear ablation on amino acid concentrations in the PVCN The rapid decreases of glutamate and aspartate concentrations in the rat caudal PVCN after cochlear ablation resemble those reported for the guinea-pig PVCN (Wenthold, 1978). The magnitude of the decrease for aspartate was also similar, but the 36% decrease of glutamate at 7 days after surgery is larger than the 27% value reported for guinea pig. This should not result from comparing caudal PVCN of rat to total PVCN of guinea pig

since the magnitude of decrease in rat did not show much rostral– caudal gradient (Fig. 3). The effects of cochlear ablation in chinchillas differed in many ways from those in rats and guinea pigs. The major similarity was that glutamate and aspartate concentrations decreased. However, the decreases were more gradual in chinchillas and reached a larger magnitude, approaching twice the rat decreases. The slower concentration decreases in chinchilla might relate to slower degeneration of the central processes of spiral ganglion cells and/or differences in metabolic reactions after destruction of their somata. The very large decreases of glutamate and aspartate concentrations would suggest that either the auditory nerve fiber contribution to glutamate and aspartate concentrations in chinchillas is much larger than in rats and guinea pigs or that, more likely, the later decreases in chinchilla include transneuronal effects on PVCN neurons as well as losses from degenerating auditory nerve fibers. There is evidence that cochlear ablation in chinchillas leads to transneuronal effects by 14 days (Morest et al., 1997), and there is evidence that many cochlear nucleus projection neurons may use glutamate and/or aspartate as a neurotransmitter (Godfrey et al., 1988; Suneja et al., 1995). If these large later decreases of aspartate and glutamate concentrations in the chinchilla PVCN relate to transneuronal effects, then such decreases apparently do not occur as rapidly or to such an extent in rats and guinea pigs (Fig. 2 and Wenthold and Gulley, 1977). The somewhat larger glutamate and aspartate decreases caudally and ventrally than rostrally and dorsally in chinchilla PVCN correlate with the dense innervation of these parts by the auditory nerve (Osen, 1970; Lorente de Nó, 1981) and suggest

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Fig. 5. Concentrations of aspartate and glutamate in subregions of the PVCN and adjacent granular region of chinchilla K. Data were assigned to subregions by comparison of sample locations in the maps of Fig. 4 with the subregion locations as defined in the atlas of Morest et al. (1990). Data are presented as means⫾S.E.M. Graphs are displayed from top to bottom as concentrations in the left PVCN and granular region, those in the right PVCN and granular region, the differences between the two sides, and the ratios of right to left side. Abbreviations for subregions are: for granular region, PX, plexiform layer; E, granule cell layer; for PVCN, AD, anterodorsal part; A, anterior part; D, dorsal part; C, central part (octopus cell region); V, ventral part. Numbers of samples are as indicated on the bars of the bottom (Ratio) graphs. They are printed on the bars of the other graphs only where they differ from the values printed on the Ratio graphs. Statistically significant differences from the averages for the PVCN central part are indicated as follows: * P⬍0.01, ** P⬍0.001, *** P⬍0.0001.

somewhat larger effects in lower- than in higher-frequency regions (Morest et al., 1997). The increases in concentration in chinchilla caudal PVCN for all amino acids at 84 days correlate at least partially with the decrease in lipid content at that time. This almost certainly results from loss of auditory nerve myelin. Previous studies have indicated that myelin in the CNS breaks down much more slowly than in the peripheral nervous system, where loss is prominent within 2 weeks (McCaman and Robins, 1959; Godfrey et al., 2004b). However, since the increases in concentration are greater for some amino acids than for others, and since there are increases in some amino acid concentrations in rat caudal PVCN at 60 days after cochlear ablation, when we did not find evidence of lipid loss, some of the increases may be associated with new growth of axons to replace lost auditory nerve innervation (Kim et al., 2004).

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The significant increase of aspartate concentration in the ipsilateral caudal PVCN up through 7 days after cochlear ablation was very surprising and not readily explained. It could represent a postsynaptic response to loss of auditory nerve input or reflect a transient metabolic phase during the degeneration of the auditory nerve fibers. Despite the close metabolic relationship between glutamate and aspartate, glutamate concentration was starting to decrease while aspartate concentration was undergoing this transient increase, suggesting that the metabolic linkage between these two amino acids, via the activity of aspartate aminotransferase, might be decreased at this early time after cochlear ablation in chinchillas, as it is in guinea-pig VCN (Wenthold, 1980). The sustained bilateral decreases in concentration of many of the amino acids in chinchilla PVCN were not found in rat. They began early, often by 3 days after cochlear ablation, and the most prominent ones, for GABA, glycine, and taurine, began earlier on the lesioned side. The effects were not larger in the chinchilla with bilateral cochlear ablation than in those with unilateral cochlear ablation. These decreases presumably represent some kind of secondary effect of the cochlear ablation in the chinchilla PVCN, both because of their bilaterality and because there is no evidence for elevated concentrations or neurotransmitter function of these amino acids in auditory nerve fibers (Fex and Wenthold, 1976; Potashner et al., 1985; Godfrey et al., 2000). In guinea pigs, unilateral cochlear ablation has been reported to result in bilateral decreases of glycine receptor binding and increases of glycine uptake in PVCN

Fig. 6. Concentrations of seven amino acids in subregions of the left (control-side) PVCN and adjacent granular region of chinchilla K. Assignment of data to subregions and other details and abbreviations are as in Fig. 5. Numbers of samples are the same for all graphs and are given in the bars of the bottom (arginine) graph.

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at mostly later times after surgery (Potashner et al., 2000) but small lesioned-side decreases, relative to contralateral, of GABA metabolic enzymes at 1 week’s survival (Fisher and Davies, 1976). Aging is another factor that can be associated with inner ear damage and changes in the central auditory system (Caspary et al., 1999). Decreases of glycine (29%) and taurine (38%) but not GABA have been reported for punched samples of cochlear nucleus in aged (29-month-old) rats as compared with young (3month-old) adults (Banay-Schwartz et al., 1989a,b). Our unpublished results indicate decreased glycine (17%, P⫽0.02) and GABA (28%, P⫽0.002) but not taurine concentrations in the caudal PVCN of aged (33-month-old) rats as compared with young (6-month-old) adults. The unusual effects of bilateral cochlear ablation surgery that was incomplete on one side, in the chinchilla with 30 days’ survival, may suggest that partial cochlear damage affects the cochlear nucleus in ways not easily predictable from the effects of complete ablation, but only tentative conclusions can be drawn from the findings in this one animal. Comparison of cochlear ablation effects to carboplatin effects in chinchilla PVCN Although the decreases of glutamate and aspartate concentrations in chinchilla caudal PVCN following cochlear ablation were slower than those in rat or guinea pig, they were faster than after carboplatin treatment (Godfrey et al., 2005). Carboplatin destroys inner hair cells and type I spiral ganglion cells in chinchillas, but the amount of destruction gradually increases over time (Ding et al., 2002; Godfrey et al., 2005), whereas cochlear ablation destroys all hair cells and spiral ganglion cells immediately. The decline of glutamate concentration in the caudal PVCN included little change at 29 days and 40% decrease at 85 days after carboplatin treatment, whereas the glutamate concentration decreased by 20% at 7 days and 55% at 14 –15 days after cochlear ablation. Thus, the decrease of glutamate concentration in caudal PVCN was more than six times faster after cochlear ablation than after carboplatin treatment. For aspartate concentration, there was no transient increase after carboplatin treatment, unlike after cochlear ablation. A 25% decrease occurred by 29 days after carboplatin treatment, but the 48% decrease at 85 days was similar to the decrease 14 –15 days after cochlear ablation. The concentrations of most of the other amino acids changed little or possibly increased after carboplatin treatment, except for GABA concentration, which decreased by about 20% at all times after carboplatin treatment, similar in timing although less in magnitude to its decrease after cochlear ablation. Further, this GABA concentration decrease showed no correlation with the amount of hair cell damage. It was suggested that the decrease in GABA concentration after carboplatin might result from a direct effect of carboplatin on brain tissue, but the even larger decreases after cochlear ablation argue possibly for some more general effect of the trauma involved in both treatments (Demediuk et al., 1989) or for an effect mediated through damage to the terminal parts of

the olivocochlear system (Kraus and Illing, 2004; Jin et al., 2005). Alternatively, the mechanism of the GABA decrease might differ between the two treatments. There is evidence that GABAergic neurons may be particularly vulnerable to a variety of agents (Ribak, 1983). Comparison of cochlear ablation effects on amino acid concentrations to those on choline acetyltransferase (ChAT) activity in rat PVCN Our previous study found 40 –50% increases of ChAT activity in the ipsilateral PVCN 1 and 2 months after unilateral cochlear ablation in the same group of rats as used for this study (Jin et al., 2005). This contrasts with the decreases of aspartate and glutamate concentrations. Most of the other amino acids showed tendencies toward increases in concentration at 1 and/or 2 months, but most of these were smaller than the increases in ChAT activity. Thus, while cholinergic elements appeared to be maintained during an estimated decrease in PVCN size after cochlear ablation (Jin et al., 2005), the maintenance of most amino acids may be less complete. Comparison of cochlear ablation effects on amino acid concentrations in rat PVCN to vestibular ganglionectomy effects on rat vestibular nuclei The effects of removal of auditory nerve innervation of the PVCN on its amino acid concentrations can be compared with the results of a similar experiment on the other inner ear sensory system. Removal of the vestibular nerve innervation of the vestibular nuclei by vestibular ganglionectomy resulted in decreased glutamate and aspartate concentrations in vestibular nuclei, often, as for PVCN, by 2 days after surgery (Li et al., 1996). However, except for the superior vestibular nucleus and the dorsal part of the lateral vestibular nucleus, the decreases were much smaller than in the PVCN at 7 days after surgery. In some cases, especially for aspartate concentrations, there was a tendency for increases toward control values by a month after surgery and for later contralateral decreases in some vestibular nuclei, resulting in a decrease in asymmetry of amino acid concentrations between lesioned and control sides. Such later decreases in asymmetry also occurred in the PVCN (Fig. 2), but to a lesser extent than in most vestibular nuclei. This may relate to more neural connections between the bilateral vestibular nuclear complexes than the cochlear nuclei (Epema et al., 1988; Shore et al., 1992). Overall, as for rat PVCN, there were few statistically significant changes of other amino acid concentrations, except that, as for PVCN, there were transient bilateral decreases of GABA concentrations in some nuclei.

CONCLUSIONS Our results indicate that the slowness of the decreases of glutamate and aspartate concentrations in the chinchilla PVCN after carboplatin treatment relates partially to a slower loss of these amino acids from the central terminal regions of auditory nerve fibers during their degeneration in chinchillas than in rats or guinea pigs and partially to

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slower deterioration of the central terminal regions of auditory nerve fibers after carboplatin treatment than after cochlear ablation. Our findings are consistent with evidence for glutamate as a transmitter of auditory nerve fibers (Wenthold et al., 1993; Hackney et al., 1996; Potashner et al., 1997; Godfrey et al., 2000, 2005) but also support aspartate. It is hard to separate evidence for glutamate or aspartate on the basis of our concentration measurements because of their close metabolic relationship, but the difference in shortterm effects of unilateral cochlear ablation on glutamate and aspartate concentrations in chinchilla is intriguing. Our results suggest that, while the major effects of cochlear ablation on glutamate and aspartate concentrations are similar among species of mammals, the details of the effects, as well as effects on other amino acids, may differ considerably. Thus, whereas we could predict that cochlear damage in humans leads to decreases of glutamate and aspartate concentrations in the VCN, it is difficult to predict their time course or magnitude or effects on other amino acids. Further, our results, together with those of previous studies (Potashner et al., 1997, 2000; Suneja et al., 2000), indicate that unilateral cochlear damage can have bilateral effects, even at the level of the first nucleus of the central auditory system. Acknowledgments—We are grateful to Brent Martin, D.V.M., and his staff of the Division of Laboratory Animal Medicine of the University of Toledo Health Science Campus for assistance and advice at various stages of this project. Support for this research was received from the American Tinnitus Association and the University of Toledo Foundation.

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(Accepted 14 December 2007) (Available online 1 January 2008)