AMPA Receptor Binding in Adult Guinea Pig Brain Stem Auditory Nuclei after Unilateral Cochlear Ablation

Experimental Neurology 165, 355–369 (2000) doi:10.1006/exnr.2000.7471, available online at http://www.idealibrary.com on

AMPA Receptor Binding in Adult Guinea Pig Brain Stem Auditory Nuclei after Unilateral Cochlear Ablation S. K. Suneja, S. J. Potashner, 1 and C. G. Benson 2 Department of Anatomy, University of Connecticut Health Center, Farmington, Connecticut 06030 Received December 1, 1999; accepted April 27, 2000

This study determined if an asymmetric hearing loss, due to unilateral cochlear ablation, could induce the regulation of intracellular AMPA receptors in brain stem auditory nuclei. In young adult guinea pigs, the high-affinity specific binding of [ 3H]AMPA was measured in the cochlear nucleus (CN), the superior olivary complex (SOC), and the auditory midbrain at 2–147 postlesion days. After correction for tissue shrinkage, changes in specific binding relative to that in age-matched unlesioned controls were interpreted as altered numbers and/or activity of intracellular AMPA receptors. In the CN, transient elevations and/or deficits in binding were evident in most regions, which usually recovered by 147 days. However, persistently deficient binding was evident ipsilaterally in the anterior part of the anteroventral CN (AVCNa). In the SOC, transient elevations in binding were evident at 2 days in the medial limb of the lateral superior olive (LSOmed) and the medial superior olive. Between 7 and 147 days, most SOC nuclei exhibited transient, temporally synchronized postlesion deficits in binding. However, late in the survival period, deficits persisted ipsilaterally in the LSOmed and the lateral (LSOlat) limb of the lateral superior olive. In the midbrain, transient elevations and/or deficits in binding were evident in the dorsal nucleus of the lateral lemniscus as well as in the central and dorsal nucleus of the inferior colliculus. A persistent deficit was evident in the intermediate nucleus of the lateral lemniscus. The findings implied that auditory neurons contain regulatory mechanisms that control the numbers and/or activity of intracellular AMPA receptors. Regulation was induced by cochlear nerve destruction and probably by changes in the excitation of glutamatergic neurons. Many of the regulatory changes were transient, except in the ipsilateral AVCNa and LSO, where postlesion downregulations were persistent. 1 To whom correspondence should be addressed at Department of Anatomy, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. Fax: (860) 679-1274. E-mail: [email protected]. 2 Present address: Department of Neurobiology, Duke University Medical Center, Durham, NC 27710.

The downregulation in the ipsilateral AVCNa was probably induced directly by the loss of cochlear nerve endings. However, other regulatory changes may have been induced by signals carried on pathways emerging from the ipsilateral CN and on centrifugal auditory pathways. © 2000 Academic Press Key Words: deafness; cochlear nucleus; superior olive; nuclei of the lateral lemniscus; inferior colliculus; [ 3H]AMPA binding; plasticity.

INTRODUCTION

Glutamatergic synapses, by virtue of their distribution and numbers, are likely to be crucial elements in the communication of environmental acoustic information from the cochlea to the brain and in the transfer of acoustic information in the ascending and descending pathways of the adult central auditory system. The cochlear nerve, which conveys acoustic information from the cochlea to neurons in the cochlear nucleus (CN), is thought to be glutamatergic (49, 54, 87, 88), as are several of the efferent pathways ascending from the CN (19, 75). Local and commissural pathways of the inferior colliculus (IC), as well as pathways ascending from the IC and the medial geniculate body, may be glutamatergic (64). In addition, projections descending from the auditory cerebral cortex and the inferior colliculus (11, 64) as well as some of the pathways local to the CN (49) are probably glutamatergic. Thus, in considering the transfer of information along the ascending and descending pathways in altered acoustic environments or after hearing impairment, it is difficult to model such neuronal communications without predicting that regulatory mechanisms may exercise control over the numbers and strengths of glutamatergic synapses. Demonstrations of plasticity in central auditory functions in the adult are consistent with the possibility that synapses in auditory pathways are regulated. For example, modifications in audition, induced by experience, and altered cellular physiology, induced by hearing loss, have been reported in adult subjects (12,

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15, 32, 40, 42, 59 – 62, 65, 86, 95). In addition, evidence that the numbers and strengths of synapses in brain auditory pathways are regulated has emerged from studies of hearing loss in adult animals. Cochlear damage that initially deafferented the CN subsequently induced transneuronal axonal pruning in several brain stem auditory pathways (34, 44, 56, 57), the growth of new axons and synaptic endings in the ventral CN (4, 5), and an altered distribution of CN efferents to the IC (41). Cochlear damage also induced regulatory changes in transmitter release from GABAergic and glycinergic synapses in brain auditory pathways (6, 10, 77) and altered glycine receptor expression and/or activity (74). We found that hearing loss in adult animals may alter the strength of excitatory glutamatergic synapses, as unilateral cochlear ablation induced regulatory changes in transmitter release from glutamatergic synaptic endings in several brain stem auditory nuclei (56). To investigate further, we wanted to assess whether hearing loss could induce regulatory changes in glutamate receptors. AMPA receptors, a subclass of ionotropic glutamate receptors (27, 33, 71, 90), are thought to be the predominant receptor in glutamatergic synapses in the central auditory system (2, 9, 13, 14, 31, 52, 53, 63, 69, 83, 84, 89, 90, 92, 93). In most auditory neurons, receptor subunits are distributed in the postsynaptic membrane and intracellularly near postsynaptic sites (53, 63, 69, 84). The intracellular receptors may constitute 50 –70% of the total AMPA receptor population (22, 24) and might serve as a reservoir for receptors in the postsynaptic membrane (8, 22, 73). Regulatory changes in expression, turnover, activity, or trafficking with the postsynaptic membrane might involve altered numbers and/or activities of intracellular or postsynaptic AMPA receptors. For example, in the hippocampus and striatum, the number of AMPA receptors in postsynaptic membranes was altered by neuronal activity, long-term potentiation, and depression, stress, and deafferentation (8, 36, 81, 82, 94). In contrast, auditory neurons may respond differently, as deafferentation of the CN had little effect on binding to AMPA receptors in the postsynaptic membranes of CN neurons (35). Therefore, should regulation of AMPA receptors occur in auditory neurons, it may be evident in the expression, turnover, or activity of the intracellular population. To examine the numbers and/or activity of intracellular AMPA receptors, we quantified the specific binding of [ 3H]AMPA to high-affinity AMPA receptors, which are thought to constitute the intracellular population (21, 23–25, 28, 45, 50, 72). To assess whether or not regulatory mechanisms could alter the intracellular receptor population in auditory pathways, we determined if binding was altered as a consequence of unilateral cochlear ablation, one example of an asymmetric hearing loss. During a postlesion period of 147 days, we compared the binding in the brain stem au-

ditory nuclei of lesioned animals to that in tissues of age-matched (1) unlesioned controls. The data were analyzed to determine if postlesion regulatory changes were evident and, if so, whether regulation depended on the ablated cochlear nerve or might involve other auditory pathways. Several preliminary findings were reported in abstracts (76, 78, 79). METHODS

Animal Subjects This study used English albino guinea pigs of either sex, weighing approximately 400 g. Guinea pigs become sexually mature by 56 (female) to 77 days (male) and can live 5– 6 years (26). The physiological characteristics of the auditory system are mature at birth (37). Guinea pigs were approximately 50 days of age when they underwent unilateral cochlear ablation and then survived an additional 2, 7, 31, 60, and 147 days before receptor-binding activity was measured. Unlesioned age-matched animals served as controls, as AMPA-binding activity may vary with age in some areas of the brain (1). Unilateral Cochlear Ablation Guinea pigs were anesthetized with pentobarbital (Nembutal, 32 mg/kg, ip; Abbott Laboratories, North Chicago, IL), supplemented as necessary with diazepam (Valium, 1–2 mg/kg, ip), and treated with atropine sulfate (0.05 mg/kg, ip). Surgical procedures were performed under aseptic conditions in a stereotaxic frame. To ablate one cochlea, the lateral wall of the left dorsal bulla was opened, the malleus and incus were removed with fine rongeurs, and the left cochlea was ablated mechanically (4, 54). The skin incision was closed with sutures. Postoperatively, animals received lactated Ringer (10 ml, sc) and buprenorphine (Buprinex; Rickitt and Coleman Inc., Richmond, VA) analgesic (0.05 mg/kg, sc, twice per day for 2 days). The extent of the cochlear ablation was monitored by microscopic inspection of the dissected bulla when tissues were prepared for binding studies. These and other procedures involving animals were approved by the University Animal Care Committee in accordance with federal and state policies. Receptor Binding Tissue preparation. Animals were anesthetized with pentobarbital (32 mg/kg, ip) and decapitated. The brain stem was removed quickly, frozen with dry ice, and cut into 15-␮m-thick transverse sections, using a cryostat set at ⫺20°C. The sections were thawmounted onto glass slides and then dried overnight at ⫺20°C in slide boxes containing silica gel desiccant (Sigma, St. Louis, MO).

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[ 3H]AMPA binding. The binding procedure was adapted from that described by Nielsen et al. (46). Each execution of the binding, autoradiography and quantification procedures described below measured the specific binding activity at one survival time in sections taken from one lesioned animal and its age-matched unlesioned control. In the binding procedure, the frozen slides were kept at room temperature for 2–3 min, preincubated (2 ⫻ 15 min, 2°C) in 50 mM Tris–HCl containing 2.5 mM CaCl 2, pH 7.4 (TBC), to remove endogenous amino acids, and then dried quickly with cold air. To measure total binding, the slides were incubated in Coplin jars containing 20 nM [ 3H]AMPA (45–53 Ci/mmol; New England Nuclear, Boston, MA) in TBC (30 min, on ice). Nonspecific binding was determined using a second set of slides containing adjacent sections, which was incubated in the solution above that also contained 1 mM L-glutamate (monoammonium salt). After incubation, all of the slides were washed by quickly dipping in a series of three jars containing ice-cold TBC, followed by a final dip in icecold 2.5% glutaraldehyde in acetone. Slides were first dried in hot air and then dried overnight at 4°C in a box containing desiccant. Autoradiography and staining. Slides and calibrated autoradiographic 3H microscales (Amersham, Arlington Heights, IL) were apposed to 3H-sensitive film (Hyperfilm- 3H; Amersham) in a light-proof Hypercassette (Amersham) for 28 days at 4°C, as described previously (74). After developing the exposed films, the slides were stained with cresyl violet and examined with a light microscope. Quantification of binding. Binding activity was quantified by densitometric analysis of the autoradiographic images on the exposed films (e.g., Fig. 3), using MicroComputer imaging device software (M4, V1.7; Imaging Research Inc., Brock University, St. Catharines, Ontario, Canada), as described previously by Suneja et al. (74). Autoradiographic images of the following auditory nuclei were identified, using the cresyl-violet-stained sections as guides, and analyzed on both sides of the brain stem: the molecular (DCNm), fusiform cell (DCNf), and deep (DCNd) layers of the dorsal cochlear nucleus (DCN); the posteroventral cochlear nucleus (PVCN); the small cell shell on the dorsal surface of the PVCN (SSpv); the caudal (AVCNc), rostral (AVCNr), and anterior part (AVCNa) of the anteroventral cochlear nucleus (AVCN); the small cell shell on the lateral surface of the AVCN (SSav); the lateral (LSOlat) and medial (LSOmed) limbs of the lateral superior olive (LSO); the medial superior olive (MSO); the medial (MNTB) and ventral (VNTB) nuclei of the trapezoid body; the ventral (VNLL), intermediate (INLL), and dorsal (DNLL) nuclei of the lateral lemniscus (NLL); and the central nucleus (ICc), external cortex (ICe), and dorsal cortex

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FIG. 1. AMPA binding in the sampled areas of the CN, SOC, NLL, and IC. The mean specific binding activities ⫹ SEM (9 –20 sections) in the sampled regions of the CN, SOC, and midbrain taken from two 52-day-old intact guinea pigs were plotted as nCi/mg fresh tissue (left ordinate) and fmol/mg fresh tissue (right ordinate). The sampled nuclei taken from 52-, 57-, 81-, 110-, and 197-day-old controls exhibited the following combined mean specific binding activities, in nCi/mg, ⫾ SEM (number of sections): DCNm, 1.79 ⫾ 0.07 (54); DCNf, 4.72 ⫾ 0.11 (103); DCNd, 1.32 ⫾ 0.05 (83); PVCN, 1.12 ⫾ 0.05 (89); AVCNc, 1.01 ⫾ 0.05 (82); AVCNr, 1.34 ⫾ 0.06 (104); AVCNa, 2.30 ⫾ 0.09 (66); SSpv, 3.89 ⫾ 0.09 (73); SSav, 3.59 ⫾ 0.11 (80); LSOlat, 1.12 ⫾ 0.05 (80); LSOmed, 1.28 ⫾ 0.05 (78); MSO, 1.44 ⫾ 0.05 (90); MNTB, 1.24 ⫾ 0.06 (77); VNTB, 1.42 ⫾ 0.06 (75); VNLL, 1.71 ⫾ 0.05 (112); INLL, 1.65 ⫾ 0.07 (73); DNLL, 1.40 ⫾ 0.06 (85); ICc, 2.76 ⫾ 0.10 (99); ICe, 3.63 ⫾ 0.10 (103); and ICd, 7.12 ⫾ 0.18 (107).

(ICd) of the inferior colliculus (IC). Specific binding values for each nucleus were typically provided by 3–13 adjacent sections per autoradiographic film. Specific binding was measured in several animals at each survival time. For example, at 2 postlesion days, binding was measured using 9 –20 sections from two unlesioned age-matched controls and 6 –22 sections from two lesioned animals. In the controls, the specific binding values for each analyzed nucleus were expressed as nanocuries per milligram of fresh tissue equivalent and averaged, and the mean was plotted as in Fig. 1. However, each individual execution of the binding protocol, which processed tissue sections from one control animal, produced a somewhat different level of binding activity. This variability was evident when comparing the binding activities in controls of the same age. Therefore, the binding activities of the controls were not compared as a function of age. In addition, it was noted that several of the analyzed nuclei or regions contained uneven distributions of binding activity. Therefore, to avoid biases in representing binding activities, we analyzed the entire contents of each nucleus or region to obtain the specific binding value. In each lesioned animal, the specific binding values for each nucleus were expressed as a percentage of those in its age-matched control, which

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was processed in the same execution of the binding protocol. All the percentage values at each survival time were averaged to provide a mean value. Similarly, mean percentage values represented the binding activity measured in 6 –22 sections from two lesioned animals at each survival time of 7, 31, and 60 days and 4 that survived 147 days. These mean percentage values were plotted as black bars in Figs. 4 – 6. At each survival time, the mean percentage value for an individual nucleus typically was calculated using nearly equal amounts of data from each contributing animal. The range in the number of tissue sections reflects the fact that some nuclei appeared in relatively few sections while others were evident in a greater number of sections. Corrections for altered tissue areas. Using the cresyl-violet-stained sections, the areas of the CN subdivisions, LSO, MSO, MNTB, VNTB, NLL, and IC were measured at each survival time in control and lesioned animals, as described previously by Suneja et al. (74). At each survival time, a mean area value was computed for each nucleus and divided by that from the age-matched controls. The resulting value, called the “area factor,” was plotted in Fig. 2. In several of the auditory nuclei, where unilateral cochlear ablation induced significant shrinkage (Fig. 2A), the mean specific binding values were corrected by multiplying them by the appropriate area factor. These corrected specific binding values were plotted in Figs. 4 and 5 as white bars. Statistical Analyses The data were analyzed using multifactor analyses of variance (ANOVA) (SPSS V6.1, Chicago, IL). Significant main effects and interactions were analyzed further, using Duncan’s post hoc multiple comparison test or Student’s t test. RESULTS Assessment of the Unilateral Cochlear Ablation

In a previous study (4), microscopic comparisons were made using sections of the temporal bone from unlesioned controls and animals that received a unilateral cochlear ablation. The ablation procedure destroyed the cochlea completely, including the bony cochlea, the organ of Corti, the spiral ganglion, and the cochlear nerve. In the present study, the cochlear ablation was assessed by microscopic inspection of the dissected left bulla before binding was measured. Postlesion Shrinkage of Auditory Nuclei

The areas of the sampled nuclei on both sides of the brain stem were measured at each survival time in lesioned and control animals. Unilateral cochlear ab-

lation resulted in significant shrinkage in the ipsilateral PVCN, AVCN, and LSO, and in the contralateral MNTB (Fig. 2A). In the ipsilateral PVCN and AVCN, shrinkage was first apparent at 31 days and progressed to 45– 49% by 147 days. In the ipsilateral LSO, shrinkage became apparent only at 147 days and amounted to 21%. In the contralateral MNTB, shrinkage was evident at 60 days and progressed to 22% by 147 days. By contrast, changes were not apparent in the size of the DCN, the small cell shell, the MSO, the VNTB, the NLL, and the IC (Fig. 2B). Postlesion shrinkage of the magnitudes reported above would concentrate receptors spatially as the amount of tissue between receptors declined. Thus, the radioactivity bound to receptors would be concentrated into a smaller area, providing a greater autoradiographic optical density. Since each [ 3H]AMPA binding value was derived from the average autoradiographic optical density within a nucleus, and since this measure took no account of tissue area (38), the optical density values, and thus the specific binding values, could be inflated by tissue shrinkage. Therefore, in nuclei that exhibited postlesion shrinkage (Fig. 2A), the binding that was uncorrected for shrinkage reflected the sum of changes in the spatial density of the binding sites plus any changes in the specific binding activity. These uncorrected measures are represented in Figs. 4 – 6 by the black bars. By contrast, when corrected for postlesion shrinkage, the measurements represented only the changes in the specific binding activity. These corrected measures are represented in Figs. 4 and 5 by the white bars. Distribution of [ 3H]AMPA Binding Activity

The sampled auditory nuclei of unlesioned controls (Fig. 1) and of lesioned animals (Fig. 3) exhibited different levels of binding activity. Statistical analysis of the specific binding activities from the 52-day-old control animals (Fig. 1) indicated the following differences (Duncan, P ⱕ 0.05). In the DCN, binding in the DCNf exceeded that in the other layers (also Fig. 3A). In the ventral cochlear neucleus, binding in the PVCN and AVCNc was similar. However, it increased in the AVCNr and increased further in the AVCNa. Binding in the small cell shell areas exceeded that in the PVCN and AVCN. A gradient of binding density was evident in the AVCN (also Fig. 3B, contralateral A) which indicated a higher density of binding dorsally, in regions representing higher frequencies of sound. In the superior olivary complex (SOC), binding activity was similar among the sampled nuclei. In the midbrain, binding in the VNLL and INLL, although similar, exceeded that in the DNLL, but was less than that in each part of the IC. The binding in the ICd exceeded that in the ICe and ICc (also Fig. 3C). The relative levels of binding activity, evident in Fig. 1 for 52-day-

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FIG. 2. Effects of unilateral cochlear ablation on the areas of the brain stem auditory nuclei. The plotted data are means ⫹ SEM of the “area factor,” which was defined as the area of a nucleus after cochlear ablation divided by the mean area of the same nucleus in the age-matched unlesioned controls. In the lesioned animals, areas were measured in sections of the PVCN (n, 6 –26), AVCN (n, 13– 40), LSO (n, 7–28), MNTB (n, 12–28), DCN (n, 6 –17), SS (n, 16 –56), MSO (n, 14 – 42), VNTB (n, 8 –24), NLL (n, 12– 47), and IC (n, 6 –15). Control animals, including all of the survival times, exhibited the following mean areas in mm 2 ⫾ SEM (number of sections): PVCN, 1.06 ⫾ 0.025 (71); AVCN, 0.99 ⫾ 0.028 (117); LSO, 0.59 ⫾ 0.016 (69); MNTB, 0.19 ⫾ 0.005 (79); DCN, 1.46 ⫾ 0.029 (69); SS, 0.418 ⫾ 0.012 (181); MSO, 0.067 ⫾ 0.002 (102); VNTB, 0.14 ⫾ .005 (73); NLL, 0.92 ⫾ 0.022 (62); and IC, 13.30 ⫾ 0.18 (57). The SS value represents summed areas of the SSpv and SSav, and the NLL value represents summed areas of the VNLL and INLL. Asterisks denote differences from the controls that were significant at the P ⱕ 0.05 level, using Student’s t test.

old controls, persisted in control animals of greater age (not shown). Effects of Cochlear Ablation on [ 3H]AMPA Binding

The specific binding of [ 3H]AMPA was measured in the sampled auditory nuclei 2, 7, 31, 60, and 147 days after unilateral cochlear ablation (Figs. 4 – 6). Inspec-

tion and statistical analysis of the data suggested that this lesion induced significant changes in binding (ANOVA, F 1,3311 ⫽ 8.94, P ⱕ 0.003) that developed as a function of survival time (ANOVA, F 4,3311 ⫽ 6.53, P ⱕ 0.001). Changes in binding were noted both on the side of the brain ipsilateral to the ablated cochlea and on the contralateral side of the brain, adjacent to

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FIG. 3. [ 3H]AMPA binding in the CN, SOC, and midbrain 147 days after unilateral cochlear ablation. (A) An autoradiograph showing typical binding in the DCN and PVCN; (B) illustrates binding in the AVCN and SOC; and (C) binding in the midbrain. Abbreviations: D, DCN; P, PVCN; A, AVCN; Sa, SSav; LL, LSOlat; ML, LSOmed; M, MSO; T, MNTB; V, VNTB; I, INLL. Bar, 1 mm.

the intact cochlea. We have designated brain structures on the ablated side as “ipsilateral” nuclei and those on the intact side as “contralateral.” Finally, the postlesion pattern of change as a function of survival time (which we refer to as the “response”) differed in the CN, the SOC, and the midbrain. CN Responses Specific binding activity was measured in the DCN, PVCN, AVCN, and the small cell shell. Most postlesion

responses represented transient changes in specific binding (Fig. 4). For example, the responses in the DCN, the small cell shell and the PVCN were roughly similar, consisting of elevations in binding during the first postlesion week and depressions at 31 and/or 60 days, followed by a return of binding to control levels (Fig. 4). By contrast, binding in much of the AVCN was transiently deficient during the first week and elevated contralaterally by 147 days. In addition, binding in the ipsilateral AVCNa exhibited a persistent decline. DCN. The first postlesion week was characterized by several transient elevations in specific binding. Binding was elevated above the controls by 37 and 127% in the contralateral DCNm and DCNf at 2 days and by 58 – 60% in the DCNd bilaterally at 7 days (Fig. 4). These elevations were succeeded by several transient deficiencies in binding at 31 or 60 days on the ipsilateral side. Binding declined by 12% in the ipsilateral DCNf at 31 days and by 17% in the ipsilateral DCNd at 60 days. By 147 days, binding activities were near control levels. SSpv and SSav. The responses in sampled areas of the small cell shell resembled those in the superficial regions of the DCN, the DCNm, and DCNf. During the first postlesion week, binding was elevated transiently by 23% in the contralateral SSpv at 2 days (Fig. 4). Binding declined transiently by 23% in the ipsilateral SSpv at 31 days and by 22–24% in the SSav bilaterally at 31 and 60 days. By 147 days, binding activities had returned to control levels. PVCN. On the ipsilateral side, without correction for postlesion shrinkage of the tissue, elevations in binding were apparent at 7, 60, and 147 days (Fig. 3A, P; Fig. 4, PVCN, black bars). After correction for shrinkage, the elevation of binding above the controls by 88% at 7 days remained. However, the elevations at 60 and 147 days were no longer evident (Fig. 4, PVCN, white bars), suggesting that they stemmed from changes in the spatial density of the binding sites. After correction for postlesion shrinkage, the responses resembled those in the deep region of the DCN, the DCNd. During the first postlesion week, binding was elevated above the controls by 78 – 88% bilaterally at 7 days (Fig. 4, PVCN black bars). Binding declined by 19% on the ipsilateral side at 60 days (Fig. 4, PVCN, white bars) and binding was near control levels by 147 days. AVCN. In each of the sampled regions of the ipsilateral AVCN, before correction for postlesion shrinkage, elevations in binding were apparent at 31–147 days (Fig. 4, AVCNc, AVCNr, AVCNa, black bars). After correction for shrinkage, these elevations were no longer evident (Fig. 4, AVCNc, AVCNr, AVCNa, white bars), suggesting that they stemmed from changes in the spatial density of the binding sites.

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FIG. 4. [ 3H]AMPA binding in the CN after unilateral cochlear ablation. Specific binding activities in the lesioned animals are plotted at each survival time as percentages of those from their age-matched controls. Black bars represent the means ⫹ SEM of the specific binding activities without correction for shrinkage. White bars represent the means ⫹ SEM of the specific binding activities corrected for shrinkage. Bars typically represent the mean of specific binding activities from 6 –22 sections. Typically, 3– 8 sections were analyzed from each animal. The number of animals per survival time appears under Methods. Asterisks denote differences between the present means and those from the appropriate age-matched controls that were significant at the P ⱕ 0.05 level, using Student’s t test. Abbreviations: ipsi, ipsilateral; contra, contralateral.

After correction for postlesion shrinkage, binding in the AVCNc was unchanged (Fig. 4, AVCNc). In the AVCNr, binding decreased by 36% ipsilaterally and by 24% contralaterally at 7 days, but subsequently returned to control levels (Fig. 4, AVCNr). Contralaterally, binding became elevated above the controls by 26% at 147 days (Fig. 3B, A; Fig. 4, AVCNr). In the ipsilateral AVCNa, binding declined from 7 to 147 days, reaching a final deficit of 33% (Fig. 4, AVCNa). Contralaterally, binding remained near control levels until 147 days, when it became elevated by 38%.

SOC Responses Specific binding was measured in the LSOlat, LSOmed, MSO, MNTB, and VNTB (Fig. 5). Most of the postlesion responses represented transient changes, except those in the ipsilateral LSOlat and LSOmed, where deficiencies in binding persisted late in the survival period. In addition, a temporal synchrony in the responses of the nuclei appeared subsequent to 2 days. Temporal synchrony was not apparent at 2 days, when binding differed among the nuclei (Duncan, P ⱕ

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FIG. 5. [ 3H]AMPA binding in the SOC after unilateral cochlear ablation. Specific binding activities in the lesioned animals are plotted at each survival time as percentages of those from their age-matched controls. Black bars represent the means ⫹ SEM of the specific binding activities without correction for shrinkage. White bars represent the means ⫹ SEM of the specific binding activities corrected for shrinkage. Bars typically represent the mean of specific binding activities from 6 –19 sections. Typically, 3– 6 sections were analyzed from each animal. The number of animals per survival time appears under Methods. Asterisks denote differences between the present means and those from the appropriate age-matched controls that were significant at the P ⱕ 0.05 level, using Student’s t test. Abbreviations: ipsi, ipsilateral; contra, contralateral.

0.05). For example, binding was decreased by 19% in the ipsilateral LSOlat, increased by 30 –50% bilaterally in the LSOmed and MSO and remained near control levels in the other nuclei (Fig. 5). However, synchrony was evident between 7 and 147 days. After correction for tissue shrinkage (Fig. 5, white bars), the responses of the ipsilateral nuclei, together with that of the contralateral MNTB, consisted of two successive cycles of deficit followed by recovery. In all the ipsilateral nuclei except the MSO, deficits of 32– 45% were evident at 7 days and were followed by deficits of 36 – 40% at 60 days. Each of these deficits was followed by a return of binding to control levels. Although the pattern of the response in the ipsilateral MSO resembled that in other ipsilateral nuclei, the deficiencies in the ipsilateral MSO at 7 and 60 days were less severe (Duncan, P ⱕ 0.05), reaching deficits of only 18 –25%. Exceptions to the pattern consisted of incomplete recovery to control levels in the ipsilateral LSOlat at 31 days as well as in the ipsilateral LSOlat and LSOmed at 147 days. In the LSOlat and LSOmed at 147 days, binding remained deficient

by 21–22%. On the contralateral side, the responses of the LSOlat, LSOmed and VNTB were similar to one another between 7 and 147 days (ANOVA, F 3,178 ⫽ 0.473, P ⫽ 0.701). These contralateral responses typically consisted of a deficit of 21– 40% at 7 days followed by a recovery to control levels. However, binding in the contralateral MSO was not deficient, but remained near control levels after 2 days. Midbrain Responses Specific binding was measured in the NLL and in subdivisions of the IC (Fig. 6). Most of the responses represented transient changes in binding that were similar on the ipsi- and contralateral sides (Duncan, P ⬎ 0.05). However, the response of the ipsilateral INLL was an exception (Duncan, P ⱕ 0.05), as it contained a persistent deficiency in binding. NLL. In the VNLL, binding remained near the control levels, except for a decrease of 10% at 2 days ipsilaterally and an elevation above the controls by 11% at 147 days contralaterally (Fig. 6, VNLL). In the

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FIG. 6. [ 3H]AMPA binding in the midbrain after unilateral cochlear ablation. Specific binding activities in the lesioned animals are plotted at each survival time as percentages of those from their age-matched controls. The bars represent the means ⫹ SEM of the specific binding activities. Bars typically represent the mean of specific binding activities from 6 –22 sections. Typically, 3–7 sections were analyzed from each animal. The number of animals per survival time appears under Methods. Asterisks denote differences between the present means and those from the appropriate age-matched controls that were significant at the P ⱕ 0.05 level, using Student’s t test. Abbreviations: ipsi, ipsilateral; contra, contralateral.

ipsilateral INLL, binding was depressed by 42% at 7 days and then recovered partially, remaining deficient by 12% at 147 days (Fig. 6, INLL). Contralaterally, the binding remained near the controls, except for an elevation above the controls by 18% at 147 days. In the ipsilateral DNLL, binding remained near the controls (Fig. 6, DNLL). Contralaterally, binding was elevated by 42% at 2 days, deficient by 28% at 7 days, and near the controls subsequently. IC. The responses of the ICc and ICd were similar to one another. For example, in these two subdivisions (Fig. 6, ICc and ICd), binding was decreased contralaterally by 8 –17% at 2 days, decreased ipsilaterally by 12–24% at 31 days, and increased bilaterally by 16 – 29% at 60 days. Binding had returned to control levels by 147 days. In the ICe, the binding remained near the controls, except for a 10% deficit contralaterally at 147 days (Fig. 6, ICe). DISCUSSION

The specific binding activity measured in the present study probably reflects the numbers and/or the activity

of intracellular AMPA receptors. The changes in binding evident after unilateral cochlear ablation were consistent with the following conclusions. First, the intracellular population of AMPA receptors in many of the sampled auditory nuclei could be altered by mechanisms that regulated AMPA receptor numbers and/or activity. Second intracellular AMPA receptor numbers and/or activity depended on the cochlear nerve that had been destroyed by the lesion and on the level of excitation of glutamatergic neurons, but that dependence was only partial. Third, postlesion changes in receptor numbers and/or activity in most of the sampled nuclei were transient, but persistent downregulations in the ipsilateral AVCNa and LSO probably represented permanent changes. Finally, in the ipsilateral AVCNa, the downregulations in receptor numbers and/or activity were probably induced directly by the loss of cochlear nerve endings. However, in other nuclei, the effects of cochlear nerve loss were likely to have been mediated by signals carried on CN pathways emerging from the ipsilateral CN and on centrifugal auditory pathways.

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[ 3H]AMPA Binding to AMPA Receptors AMPA receptors are thought to be heterooligomeric macromolecules assembled from four subunits, GluR1– GluR4, each of which can be expressed as two splice variants, termed “flip” and “flop” (27, 33, 71, 90). AMPA receptors are probably distributed in intracellular and synaptic sites, as receptor subunit immunoreactivity has been found in the cytoplasm of neurons and in the postsynaptic density of junctions exhibiting the morphology of excitatory synapses (47, 53, 63, 69, 84). At most of the synaptic junctions that have been examined, including those in auditory nuclei, AMPA receptor subunit immunoreactivity was localized postsynaptically. However, a proportion of the synaptic junctions in the LSO exhibited subunit immunoreactivity associated with presynaptic membranes (69). The trafficking of receptors between the intracellular population and the pre- and postsynaptic membranes is poorly understood, although the redistribution of receptors between these sites might contribute to normal activity and to plasticity (8, 22, 73). The intracellular receptors have been estimated to comprise approximately 50 – 70% of the total receptor population (22, 24) and to bind [ 3H]AMPA with relatively high affinity, having a K d in the 5–20 nM range (21, 23–25, 28, 45, 50, 72, 73). The remaining receptors bind [ 3H]AMPA with lower affinity, exhibiting a K d in the 500 –1000 nM range, and are thought to be in postsynaptic membranes. Therefore, the present measurements are likely to represent binding mainly to the intracellular receptors, as we employed a concentration of [ 3H]AMPA (20 nM) that was near the K d of the high-affinity binding. As indicated by the specific binding of [ 3H]AMPA, presumed intracellular AMPA receptors were present in each of the sampled auditory nuclei in unlesioned guinea pigs (Fig. 1). A gradient of AMPA receptor density was apparent in the AVCN (Fig. 3B, A) and was consistent with greater receptor densities in regions representing the higher frequencies of sound. The distribution of binding activity among the sampled auditory nuclei was roughly similar to those of GluR subunit mRNA expression and GluR subunit immunostaining (53, 66), suggesting that high-affinity binding activity reflected the intracellular receptor population. A rough similarity between the distributions of binding activity and glutamate concentration in the CN and SOC (17–19) suggested a correlation between the presence of intracellular AMPA receptors and glutamatergic presynaptic endings. However, the DCNm, PVCN, and SOC were exceptions to these concurrences, as AMPA-binding activity was low in these areas compared to the relative levels of GluR subunit mRNA, GluR subunit immunostaining, and glutamate concentration. These discrepancies might possibly stem from quenching of the radioactive emissions from bound [ 3H]AMPA due to the presence of relatively large con-

centrations of myelin in these areas or from regulation that persistently lowered the numbers and/or affinity of the intracellular receptors. Postlesion Shrinkage of Auditory Nuclei Unilateral cochlear ablation resulted in shrinkage of the ipsilateral PVCN and AVCN (Fig. 2A), which may stem mainly from the degeneration of cochlear afferents and the atrophy of neuronal somata and dendrites (4, 39, 51, 56, 70, 85). Postlesion atrophy in the ipsilateral LSO and the contralateral MNTB (Fig. 2A) might be a consequence of transneuronal degeneration, which has been observed in the SOC after cochlear damage (34, 44, 56, 57). The present atrophy resembled that in the ventral CN and SOC reported in other studies of cochlear ablation in adult animals (39, 41, 43, 51, 57, 85). However, significant neuronal loss has not been substantiated in the adult mammalian central auditory system after cochlear lesions (e.g., 30, 39, 57, but see 20, 80). The degree of shrinkage observed in the present study would concentrate AMPA receptors into smaller areas, providing greater autoradiographic densities and inflated specific binding values. Therefore, specific binding values were corrected for tissue shrinkage to eliminate the contribution of shrinkageinduced, spatial concentration of the receptors. Postlesion Changes in [ 3H]AMPA Binding The CN. The dependence of AMPA receptor numbers and/or activity on the degenerated cochlear nerve was indicated by the postlesion binding activities in the CN that differed from those of the unlesioned controls. For example, in the ipsilateral AVCNa, which receives many cochlear nerve endings (7, 29), the postlesion response was consistent with a partial but persistent downregulation of intracellular AMPA receptor numbers and/or activity (Fig. 4). Since a deficiency in binding was not apparent contralaterally, the location and the schedule of these changes resembled those of the degeneration of the lesioned cochlear nerve (4, 87). Thus, the binding activity that was lost probably depended directly on the presence of normal cochlear nerve endings. Presumably, the failure of glutamatergic synaptic transmission at cochlear nerve endings (49, 54, 87, 88) or the loss of one or more types of molecule released from these endings might constitute a signal to AVCNa cells that induced the downregulation of their AMPA receptors. This idea receives indirect support from similar studies in other brain areas, where long-term changes in AMPA-binding activity were evident after the destruction of glutamatergic afferents (58, 94). However, in the present study, approximately two-thirds of the binding activity remained, suggesting an additional unidentified influence that sustained a proportion of the intracellular receptor population.

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In the other areas of the CN that receive cochlear nerve endings, binding that was normal at 2 postlesion days, but transiently altered on both sides of the brain at 7 days, suggested that some of the effects of cochlear nerve loss might be mediated through noncochlear auditory pathways. The altered binding was consistent with elevated numbers and/or activity of intracellular AMPA receptors in the DCNd and PVCN, as well as with deficiencies in the AVCNr. Since these areas receive many cochlear nerve endings (7, 29), it is tempting to ascribe the ipsilateral upregulations to denervation supersensitivity due to the loss of one cochlear nerve. However, this hypothesis is challenged by the downregulation in the ipsilateral AVCNr as well as the bilateral and symmetrical nature of the responses in each of the tissues. Another possibility is that signals originating in the ipsilateral CN while cochlear nerve endings degenerated at 3– 4 postlesion days (4, 87) might have been carried by CN efferents to other auditory nuclei, such as the periolivary nuclei of the SOC and the IC. By 7 days, centrifugal projections from these areas (3, 67, 91) may have carried signals that induced the regulation of AMPA receptors in both the ipsi- and contralateral CN. Despite the transient changes at 7 days, the subsequent recovery and maintenance of binding activities near the control levels again suggested the presence of another influence that could sustain the receptor population. In areas of the CN that receive few cochlear nerve endings, altered binding again implied the involvement of noncochlear auditory pathways. For example, altered binding contralaterally in the DCNm, DCNf, and SSpv, at 2 postlesion days (Fig. 4), was consistent with modest transient elevations in the numbers and/or activity of intracellular AMPA receptors. Additional changes at 31– 60 days suggested modest transient deficiencies in the ipsilateral DCNf and SSpv, as well as bilaterally in the SSav, that developed approximately 27 days after the complete degeneration of the cochlear nerve endings (4, 87). Since these areas receive a significant input from centrifugal projections (3, 7, 29, 67, 91), regulatory signals that originated in the deafferented ipsilateral CN may have arrived ultimately via the centrifugal projections. The SOC. The dependence of intracellular AMPA receptor levels and/or activities in the SOC on the degenerated cochlear nerve was indicated by the presence of postlesion changes in the binding activity. Since there are no known synaptic connections of the cochlear nerve in the SOC, the signals that induced these changes are likely to have originated from CN cells and their efferents (29, 68). Three additional observations support this view. First, deficient transmitter release from glutamatergic synaptic endings in the ipsilateral CN at 2 postlesion days (56) coincided with a downregulation of receptors in the ipsilateral LSOlat

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and bilateral upregulations in the LSOmed and MSO (Fig. 5). This temporal concurrence implied that subnormal excitation of cells in the ipsilateral CN, where glutamatergic cochlear afferents were degenerating, may have induced the regulation of receptor levels or activities in the SOC. Second, deficient transmitter release from glutamatergic synaptic endings in the ipsilateral CN at 2– 8 postlesion days (54, 56) and in the SOC at 5 postlesion days (56) were accompanied by a bilateral downregulation of receptors in most of the sampled SOC nuclei at 7 days (Fig. 5). Since glutamatergic CN efferents project to the SOC (16, 19, 75), these findings implied that subnormal excitation of cells in the ipsilateral CN and subnormal excitatory transmission in CN efferents probably contributed to receptor downregulation in the SOC nuclei. Third, deficient transmitter release from glutamatergic synaptic endings in the contralateral CN at 59 postlesion days (56) coincided with downregulations of receptors in the ipsilateral SOC nuclei as well as in the contralateral LSOmed and MNTB at 60 days (Fig. 5). Moreover, recovery of transmitter release from glutamatergic synaptic endings in the ipsi- and the contralateral CN between 59 and 145 postlesion days (56) coincided with the recovery of binding activities in the SOC (Fig. 5). This implied that subnormal excitation of cells in the contralateral CN, where cochlear afferents remained intact, could also induce downregulatory changes in the SOC. In addition, recovery of normal excitation of CN cells could induce upregulatory changes in the SOC. Taken together, this evidence implies that the synchronous pattern of deficit followed by recovery of AMPA binding in the SOC may have been induced by changes in the level of glutamatergic excitation of CN cells on either side of the brain and changes in the level of transmission from CN efferents. The responses in the MSO were an exception to this pattern, as postlesion deficiencies in binding were modest or absent (Fig. 5). Although this might theoretically reflect a different level of signaling from CN efferents or a reduced capacity of MSO cells to respond, it is more likely to stem from rather dramatic structural changes in the synaptic connections of the MSO that appear soon after cochlear ablation. For example, after unilateral cochlear damage, synaptic endings disappeared from the half of each MSO ipsilateral to the ablated cochlear nerve, while synaptic endings in the contralateral half of each MSO appeared to increase in density and the synaptic field occupied a larger area (44, 55). These changes began by 7 postlesion days and were frankly evident at 147 days. Since many synapses in the MSO are probably glutamatergic (19, 75), the apparent growth of new synapses might have increased the expression of AMPA receptors and suppressed or masked any downregulatory activity. The midbrain. The dependence of intracellular

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AMPA receptor levels and/or activities in the midbrain on the degenerated cochlear nerve was indicated by the presence of postlesion changes in the binding activity, which were most evident in the INLL, DNLL, ICc, and ICd. Since there are no known synaptic connections of the cochlear nerve in these nuclei, the changes in binding are likely to have been induced by signals from CN cells and their efferents (29, 48, 68). For example, in the ipsilateral INLL and the contralateral DNLL, the altered binding during the first postlesion week, which culminated in receptor downregulation by 7 days (Fig. 6), coincided with deficiencies in transmitter release from glutamatergic synaptic endings in the ipsilateral CN at 2– 8 postlesion days (54, 56). In addition, the subsequent recovery of receptor levels and/or activity in the INLL and DNLL occurred during the recovery from deficiencies in release from glutamatergic synaptic endings in the ipsilateral CN (56). In the IC, however, intracellular AMPA receptor levels and/or activity might have been influenced initially by the level of excitation of cells in the ipsilateral CN, but by 60 postlesion days, the predominant influence appears to have been the level of excitation of IC cells. For example, at 2 postlesion days, deficient transmitter release from glutamatergic synaptic endings in the ipsilateral CN (56) coincided with a downregulation of receptors in the contralateral ICc and ICd (Fig. 6). This concurrence implied that subnormal excitation of cells in the ipsilateral CN may have induced regulation of receptors in the ICc and ICd, presumably by signaling through CN efferents that project to the IC (29, 48). However, at 59 postlesion days, bilateral upregulation of glutamatergic excitation of ICc cells (56) coincided with the bilateral upregulation of receptors in the ICc and ICd (Fig. 6). Since glutamatergic IC neurons are thought to project widely in the ipsi- and contralateral IC (64), they might have carried signals that induced receptor upregulation.

alterations in expression, RNA translation, posttranslational processing, receptor construction, turnover, and/or trafficking with the postsynaptic membrane, further definition of the contributing mechanisms must await additional studies. The presence of changes in the intracellular population of AMPA receptors implies that regulatory control of the receptors in postsynaptic membranes might also be possible. However, previous binding studies found little evidence of significant change in the CN after cochlear deafferentation (35), suggesting that the number of postsynaptic receptors was not altered. The explanation of this paradox may lie in further studies that determine if deafferentation can alter the subunit composition of the postsynaptic receptors. ACKNOWLEDGMENTS The authors are grateful to Ms. J. Gross for assistance with the area measurements of auditory nuclei and Ms. J. Anderson for the densitometric analysis of most of the autoradiographic films. This work was supported by grant DC00199 (S.J.P.) from the National Institute on Deafness and Other Communication Disorders.

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