Multiple Sources of Cholinergic Input to the Superior Olivary Complex.

The superior olivary complex (SOC) is a major computation center in the brainstem auditory system. Despite previous reports of high expression levels of cholinergic receptors in the SOC, few studies have addressed the functional role of acetylcholine in the region. The source of the cholinergic innervation is unknown for all but one of the nuclei of the SOC, limiting our understanding of cholinergic modulation. The medial nucleus of the trapezoid body, a key inhibitory link in monaural and binaural circuits, receives cholinergic input from other SOC nuclei and also from the pontomesencephalic tegmentum. Here, we investigate whether these same regions are sources of cholinergic input to other SOC nuclei. We also investigate whether individual cholinergic cells can send collateral projections bilaterally (i.e., into both SOCs), as has been shown at other levels of the subcortical auditory system. We injected retrograde tract tracers into the SOC in gerbils, then identified retrogradely-labeled cells that were also immunolabeled for choline acetyltransferase, a marker for cholinergic cells. We found that both the SOC and the pontomesencephalic tegmentum (PMT) send cholinergic projections into the SOC, and these projections appear to innervate all major SOC nuclei. We also observed a small cholinergic projection into the SOC from the lateral paragigantocellular nucleus of the reticular formation. These various sources likely serve different functions; e.g., the PMT has been associated with things such as arousal and sensory gating whereas the SOC may provide feedback more closely tuned to specific auditory stimuli. Further, individual cholinergic neurons in each of these regions can send branching projections into both SOCs. Such projections present an opportunity for cholinergic modulation to be coordinated across the auditory brainstem.

Quantitative distribution of choline acetyltransferase activity in rat trapezoid body.

There is evidence for a function of acetylcholine in the cochlear nucleus, primarily in a feedback, modulatory effect on auditory processing. Using a microdissection and quantitative microassay approach, choline acetyltransferase activity was mapped in the trapezoid bodies of rats, in which the activity is relatively higher than in cats or hamsters. Maps of series of sections through the trapezoid body demonstrated generally higher choline acetyltransferase activity rostrally than caudally, particularly in its portion ventral to the medial part of the spinal trigeminal tract. In the lateral part of the trapezoid body, near the cochlear nucleus, activities tended to be higher in more superficial portions than in deeper portions. Calculation of choline acetyltransferase activity in the total trapezoid body cross-section of a rat with a comprehensive trapezoid body map gave a value 3-4 times that estimated for the centrifugal labyrinthine bundle, which is mostly composed of the olivocochlear bundle, in the same rat. Comparisons with other rats suggest that the ratio may not usually be this high, but it is still consistent with our previous results suggesting that the centrifugal cholinergic innervation of the rat cochlear nucleus reaching it via a trapezoid body route is much higher than that reaching it via branches from the olivocochlear bundle. The higher choline acetyltransferase activity rostrally than caudally in the trapezoid body is consistent with evidence that the centrifugal cholinergic innervation of the cochlear nucleus derives predominantly from locations at or rostral to its anterior part, in the superior olivary complex and pontomesencephalic tegmentum.

COMT and the neurogenetic architecture of hearing loss induced tinnitus.

As the COMT polymorphism is especially prominent in the prefrontal cortex and has been associated with auditory gating, we hypothesize that tinnitus patients with this polymorphism have altered activity in the ventromedial prefrontal/anterior cingulate areas that modulates the tinnitus percept. To test this, we recruited a total of 40 tinnitus subjects and 20 healthy controls for an EEG study. A comparison between tinnitus subjects and healthy controls and their frequency of being Val/Val genotype or Met carriers (including Val/Met and Met/Met genotype) shows no significant effect, suggesting that the distributions for the tinnitus and healthy groups are similar. Our results show that an interaction between the amount of hearing loss and the COMT Val158Met polymorphism can increase susceptibility to the clinical manifestation of tinnitus. We further demonstrate that the parahippocampus becomes involved in tinnitus in patients with hearing loss that are Met carriers. In these patients, the parahippocampus sends more tinnitus information to the pregenual anterior cingulate cortex and auditory cortex that is specifically related with increased loudness. At the same time, the pregenual anterior cingulate cortex, which normally functions as a gatekeeper, is not cancelling this auditory information, ultimately leading to increased tinnitus loudness.

A complex mTOR response in habituation paradigms for a social signal in adult songbirds.

Nonassociative learning is considered simple because it depends on presentation of a single stimulus, but it likely reflects complex molecular signaling. To advance understanding of the molecular mechanisms of one form of nonassociative learning, habituation, for ethologically relevant signals we examined song recognition learning in adult zebra finches. These colonial songbirds learn the unique song of individuals, which helps establish and maintain mate and other social bonds, and informs appropriate behavioral interactions with specific birds. We leveraged prior work demonstrating behavioral habituation for individual songs, and extended the molecular framework correlated with this behavior by investigating the mechanistic Target of Rapamycin (mTOR) signaling cascade. We hypothesized that mTOR may contribute to habituation because it integrates a variety of upstream signals and enhances associative learning, and it crosstalks with another cascade previously associated with habituation, ERK/ZENK. To begin probing for a possible role for mTOR in song recognition learning, we used a combination of song playback paradigms and bidirectional dysregulation of mTORC1 activation. We found that mTOR demonstrates the molecular signatures of a habituation mechanism, and that its manipulation reveals the complexity of processes that may be invoked during nonassociative learning. These results thus expand the molecular targets for habituation studies and raise new questions about neural processing of complex natural signals.

Effects of surgical lesions on choline acetyltransferase activity in the cat cochlea.

Although it is well established that the choline acetyltransferase (ChAT, the enzyme for acetylcholine synthesis) in the mammalian cochlea is associated with its olivocochlear innervation, the distribution of this innervation in the cochlea varies somewhat among mammalian species. The quantitative distribution of ChAT activity in the cochlea has been reported for guinea pigs and rats. The present study reports the distribution of ChAT activity within the organ of Corti among the three turns of the cat cochlea and the effects of removing olivocochlear innervation either by a lateral cut aimed to totally transect the left olivocochlear bundle or a more medial cut additionally damaging the superior olivary complex on the same side. Similarly to results for guinea pig and rat, the distribution of ChAT activity in the cat outer hair cell region showed a decrease from base to apex, but, unlike in the guinea pig and rat, the cat inner hair cell region did not. As in the rat, little ChAT activity was measured in the outer supporting cell region. As previously reported for whole cat cochlea and for rat cochlear regions, transection of the olivocochlear bundle resulted in almost total loss of ChAT activity in the hair cell regions of the cat cochlea. Lesions of the superior olivary complex resulted in loss of ChAT activity in the inner hair cell region of all cochlear turns only on the lesion side but bilateral losses in the outer hair cell region of all turns. The results are consistent with previous evidence that virtually all cholinergic synapses in the mammalian cochlea are associated with its olivocochlear innervation, that the olivocochlear innervation to the inner hair cell region is predominantly ipsilateral, and that the olivocochlear innervation to the outer hair cells is bilateral.

Differential patterns of histone methylase EHMT2 and its catalyzed histone modifications H3K9me1 and H3K9me2 during maturation of central auditory system.

Histone methylation is an important epigenetic mark leading to changes in DNA accessibility and transcription. Here, we investigate immunoreactivity against the euchromatic histone-lysine N-methyltransferase EHMT2 and its catalyzed mono- and dimethylation marks at histone 3 lysine 9 (H3K9me1 and H3K9me2) during postnatal differentiation of the mouse central auditory system. In the brainstem, expression of EHMT2 was high in the first postnatal week and down-regulated thereafter. In contrast, immunoreactivity in the auditory cortex (AC) remained high during the first year of life. This difference might be related to distinct demands for adult plasticity. Analyses of two deaf mouse models, namely Cldn14 (-/-) and Cacna1d (-/-), demonstrated that sound-driven or spontaneous activity had no influence on EHMT2 immunoreactivity. The methylation marks H3K9me1 and H3K9me2 were high throughout the auditory system up to 1 year. Young auditory neurons showed immunoreactivity against both methylations at similar intensities, whereas many mature neurons showed stronger labeling for either H3K9me1 or H3K9me2. These differences were only poorly correlated with cell types. To identify methyltransferases contributing to the persistent H3K9me1 and H3K9me2 marks in the adult brainstem, EHMT1 and the retinoblastoma-interacting zinc-finger protein RIZ1 were analyzed. Both were down-regulated during brainstem development, similar to EHMT2. Contrary to EHMT2, EHMT1 was also down-regulated in adult cortical areas. Together, our data reveal a marked difference in EHMT2 levels between mature brainstem and cortical areas and a decoupling between EHMT2 abundance and histone 3 lysine 9 methylations during brainstem differentiation. Furthermore, EHMT1 and EHMT2 are differentially expressed in cortical areas.

Choline acetyltransferase activity in the hamster central auditory system and long-term effects of intense tone exposure.

Acoustic trauma often leads to loss of hearing of environmental sounds, tinnitus, in which a monotonous sound not actually present is heard, and/or hyperacusis, in which there is an abnormal sensitivity to sound. Research on hamsters has documented physiological effects of exposure to intense tones, including increased spontaneous neural activity in the dorsal cochlear nucleus. Such physiological changes should be accompanied by chemical changes, and those chemical changes associated with chronic effects should be present at long times after the intense sound exposure. Using a microdissection mapping procedure combined with a radiometric microassay, we have measured activities of choline acetyltransferase (ChAT), the enzyme responsible for synthesis of the neurotransmitter acetylcholine, in the cochlear nucleus, superior olive, inferior colliculus, and auditory cortex of hamsters 5 months after exposure to an intense tone compared with control hamsters of the same age. In control hamsters, ChAT activities in auditory regions were never more than one-tenth of the ChAT activity in the facial nerve root, a bundle of myelinated cholinergic axons, in agreement with a modulatory rather than a dominant role of acetylcholine in hearing. Within auditory regions, relatively higher activities were found in granular regions of the cochlear nucleus, dorsal parts of the superior olive, and auditory cortex. In intense-tone-exposed hamsters, ChAT activities were significantly increased in the anteroventral cochlear nucleus granular region and the lateral superior olivary nucleus. This is consistent with some chronic upregulation of the cholinergic olivocochlear system influence on the cochlear nucleus after acoustic trauma.

Core-and-belt organisation of the mesencephalic and forebrain auditory centres in turtles: expression of calcium-binding proteins and metabolic activity.

The distribution of immunoreactivity to the calcium-binding proteins parvalbumin, calbindin and calretinin and of cytochrome oxidase activity was studied in the mesencephalic (torus semicircularis), thalamic (nucleus reuniens) and telencephalic (ventromedial part of the anterior dorsal ventricular ridge) auditory centres of two chelonian species Emys orbicularis and Testudo horsfieldi. In the torus semicircularis, the central nucleus (core) showed intense parvalbumin immunoreactivity and high cytochrome oxidase activity, whereas the laminar nucleus (belt) showed low cytochrome oxidase activity and dense calbindin/calretinin immunoreactivity. Within the central nucleus, the central and peripheral areas could be distinguished by a higher density of parvalbumin immunoreactivity and cytochrome oxidase activity in the core than in the peripheral area. In the nucleus reuniens, the dorsal and ventromedial (core) regions showed high cytochrome oxidase activity and immunoreactivity to all three calcium-binding proteins, while its ventrolateral part (belt) was weakly immunoreactive and showed lower cytochrome oxidase activity. In the telencephalic auditory centre, on the other hand, no particular region differed in either immunoreactivity or cytochrome oxidase activity. Our findings provide additional arguments in favour of the hypothesis of a core-and-belt organisation of the auditory sensory centres in non-mammalian amniotes though this organisation is less evident in higher order centres. The data are discussed in terms of the evolution of the auditory system in amniotes.

Estrogen receptors in the central auditory system of male and female mice.

The estrogen receptors in the central auditory system of male and female mice were characterized using immunocytochemical methods. Estrogen receptors alpha and beta (ERalpha, ERbeta) were localized predominantly in the ventral cochlear nucleus, nucleus of the trapezoid body, the lateral- and medio-ventral periolivary nuclei, the dorsal lateral lemniscus, and the inferior colliculus. The medial geniculate nucleus was negative for both ERalpha and ERbeta whereas the auditory cortex was positive for ERalpha. The lateral superior olive, the ventral lateral lemniscus and the central nucleus of the inferior colliculus expressed only ERbeta. The differential localization of ERalpha and ERbeta may indicate distinct roles for these two receptors in auditory processing. No major differences in the pattern, number or intensity of receptor expression was found between male and female animals. The comprehensive anatomic map that is constructed for ERalpha and ERbeta in the central auditory pathway will be a useful foundation to elucidate the complexity of estrogen actions in the auditory system.

Afferents to the cochlear nuclei and nucleus laminaris from the ventral nucleus of the lateral lemniscus in the zebra finch (Taeniopygia guttata).

The presence and nature of a descending projection from the ventral nucleus of the lateral lemniscus (LLV) to the cochlear nuclei (NA, NM) and the third-order nucleus laminaris (NL) was investigated in a songbird using tract tracing and GAD immunohistochemistry. Tracer injections into LLV produced anterograde label in the ipsilateral NA, NM and NL, which was found not to be GABAergic. Double retrograde labeling from LLV and NA/NM/NL ruled out the possibility that the LLV projection actually arose from collaterals of superior olivary projections to NA/NM/NL. The LLV projection may be involved in the discrimination of laterality of auditory input.

Compartment-specific regulation of plasma membrane calcium ATPase type 2 in the chick auditory brainstem.

Calcium signaling plays a role in synaptic regulation of dendritic structure, usually on the time scale of hours or days. Here we use immunocytochemistry to examine changes in expression of plasma membrane calcium ATPase type 2 (PMCA2), a high-affinity calcium efflux protein, in the chick nucleus laminaris (NL) following manipulations of synaptic inputs. Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Deprivation of the contralateral projection from NM to NL leads to rapid retraction of ventral, but not the dorsal, dendrites of NL neurons. Immunocytochemistry revealed symmetric distribution of PMCA2 in two neuropil regions of normally innervated NL. Electron microscopy confirmed that PMCA2 localizes in both NM terminals and NL dendrites. As early as 30 minutes after transection of the contralateral projection from NM to NL or unilateral cochlea removal, significant decreases in PMCA2 immunoreactivity were seen in the deprived neuropil of NL compared with the other neuropil that continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for microtubule-associated protein 2, which affects cytoskeleton stabilization. These results suggest that PMCA2 is regulated independently in ventral and dorsal NL dendrites and/or their inputs from NM in a way that is correlated with presynaptic activity. This provides a potential mechanism by which deprivation can change calcium transport that, in turn, may be important for rapid, compartment-specific dendritic remodeling.

Functional identification of sensory mechanisms required for developmental song learning.

A young male zebra finch (Taeniopygia guttata) learns to sing by copying the vocalizations of an older tutor in a process that parallels human speech acquisition. Brain pathways that control song production are well defined, but little is known about the sites and mechanisms of tutor song memorization. Here we test the hypothesis that molecular signaling in a sensory brain area outside of the song system is required for developmental song learning. Using controlled tutoring and a pharmacological inhibitor, we transiently suppressed the extracellular signal-regulated kinase signaling pathway in a portion of the auditory forebrain specifically during tutor song exposure. On maturation, treated birds produced poor copies of tutor song, whereas controls copied the tutor song effectively. Thus the foundation of normal song learning, the formation of a sensory memory of tutor song, requires a conserved molecular pathway in a brain area that is distinct from the circuit for song motor control.

Synaptic ultrastructure of Drosophila Johnston's organ axon terminals as revealed by an enhancer trap.

The role of auditory circuitry is to decipher relevant information from acoustic signals. Acoustic parameters used by different insect species vary widely. All these auditory systems, however, share a common transducer: tympanal organs as well as the Drosophila flagellar ears use chordotonal organs as the auditory mechanoreceptors. We here describe the central neural projections of the Drosophila Johnston's organ (JO). These neurons, which represent the antennal auditory organ, terminate in the antennomechanosensory center. To ensure correct identification of these terminals we made use of a beta-galactosidase-expressing transgene that labels JO neurons specifically. Analysis of these projection pathways shows that parallel JO fibers display extensive contacts, including putative gap junctions. We find that the synaptic boutons show both chemical synaptic structures as well as putative gap junctions, indicating mixed synapses, and belong largely to the divergent type, with multiple small postsynaptic processes. The ultrastructure of JO fibers and synapses may indicate an ability to process temporally discretized acoustic information.

Catecholaminergic inputs to aromatase cells in the canary auditory forebrain.

The caudomedial nidopallium in songbirds is a specialized forebrain auditory region involved in the processing of species-typical vocalizations. It receives a prominent catecholaminergic projection with many fibers forming basket-like structures around non-immunoreactive cells. We investigated in male canaries the anatomical relationship between tyrosine hydroxylase and cells immunoreactive for the steroid metabolizing enzyme, aromatase, in the caudomedial nidopallium using double-label immunocytochemistry. Fibers immunoreactive for tyrosine hydroxylase established numerous close contacts with aromatase-immunoreactive cells and often encircled these cells to form basket-like structures. Aromatase containing cells in the caudomedial nidopallium are therefore a major target of catecholaminergic inputs in canary. Interactions between catecholaminergic systems and aromatase in the caudomedial nidopallium may provide one mechanism for the regulation of estrogens involved in song perception and memorization.

A-kinase anchoring proteins in amygdala are involved in auditory fear memory.

A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that bind the regulatory subunits of protein kinase A (PKA). AKAP binding to PKA regulates the phosphorylation of various proteins, some of which have been implicated in synaptic plasticity and memory consolidation. Here we show that the regulatory subunits of PKA are colocalized with AKAP150 (an AKAP isoform that is expressed in the brain) in the lateral amygdala (LA) and that infusion to the LA of the peptide St-Ht31, which blocks PKA anchoring onto AKAPs, impairs memory consolidation of auditory fear conditioning.

Conductive hearing loss results in changes in cytochrome oxidase activity in gerbil central auditory system.

Conductive hearing loss (CHL) restricts auditory input to an intact peripheral auditory system. Effects of deprivation on the central auditory system (CAS) have been debated, although a number of studies support the hypothesis that CHL can cause modification of CAS structure and function. The present study was designed to test the hypothesis that unilateral CHL results in a decrease in cytochrome oxidase (CO) activity in CAS nuclei that receive major afferent input from the affected ear. Gerbils at postnatal day 12 (P21) or 6-8 weeks underwent left unilateral CHL (malleus removal), cochlear ablation, or a sham surgical procedure. After a survival time of 48 hours or 3 weeks, animals were sacrificed and tissue was processed for cytochrome oxidase histochemistry. Optical density (OD) measurements were made from individual neurons in the anteroventral cochlear nucleus (AVCN) and from medial and lateral dendritic fields in the medial superior olivary nucleus (MSO), the lateral superior olivary nucleus, and the inferior colliculus. The width of the CO-stained neuropil in MSO was also measured as an estimate of dendritic length. OD measures were corrected to neutral areas of the brain. Cochlear ablation caused significant decreases in CO activity in left lower brainstem nuclei, particularly in adult animals. Following CHL, a significant decrease in CO activity was observed in the ipsilateral AVCN and a significant increase was observed in the contralateral AVCN. Cochlear ablation resulted in decreased width of MSO neuropil containing dendrites that receive primary input from the ablated ear. CHL resulted in a significant increase in the width of MSO neuropil on both sides of the brain in the P21 animals that survived 3 weeks but not in P21 animals that survived only 48 hours or in the adult animals. Unilateral CHL is associated with changes in CO activity in the AVCN and may affect MSO dendritic length in younger animals.

The fine organization of nigro-collicular channels with additional observations of their relationships with acetylcholinesterase in the rat.

The nigro-collicular pathway that links the basal ganglia to the sensorimotor layers of superior colliculus plays a crucial role in promoting orienting behaviors. This connection originating in the pars reticulata and lateralis of the substantia nigra has been shown in rat and cat to be topographically organized. In rat, a functional compartmentalization of the substantia nigra has also been shown reflecting that of the striatum. In light of this, we reinvestigated the topographical arrangement of the nigro-collicular pathway by examining the innervation of each nigral functional zone. We performed small injections of either biocytin or wheatgerm agglutinin conjugated with horseradish peroxidase restricted to identified somatic, visual and auditory nigral zones. Frontally cut sections showed that innervations provided by the three main nigral zones form a mosaic of complementary domains stratified from the stratum opticum to the ventral part of the intermediate collicular layers, with the somatic afferents sandwiched between the visual and the auditory ones. When reconstructed from semi-horizontal sections, nigral innervations organized in the form of a honeycomb-like array composed of 100 cylindrical modules covering three-quarters of the collicular surface. Such a modular architecture is reminiscent of the acetylcholinesterase lattice we previously described in rat intermediate collicular layers. In the enzyme lattice, the surroundings of the cylindrical modules are composed of a mosaic of dense and diffuse enzyme subdomains. Thus, we compared the distribution of the overall nigral projection and of its constituent channels with the acetylcholinesterase lattice. The procedure combined axonal labelling with histochemistry on single sections for acetylcholinesterase activity. The results demonstrate that the overall nigral projection overlaps the acetylcholinesterase lattice and its constituent channels converge with either the dense or the diffuse enzyme subdomains. The stereometric arrangement of the nigro-collicular pathway is suggestive of an architecture promoting the selection of collicular motor programs for different classes of orienting behavior.

PMCA2 mutation causes structural changes in the auditory system in deafwaddler mice.

Homozygous deafwaddler mice (dfw/dfw) have a mutation in the gene encoding plasma membrane Ca2+ATPase isoform 2 (Pmca2). They walk with a hesitant and wobbly gait, display head bobbing and are deaf. Light microscopy and transmission electron microscopy were used to evaluate the nature and relationship of morphological changes in the cochlea, spiral ganglion cells and spherical cells of the cochlear nucleus in homozygous and heterozygous mice of different ages and controls. Ultrastructural findings showed that in 7 week old homozygous (dfw) mice, inner hair cells and their afferent terminals were present although outer hair cells appeared apoptotic. Stereocilia were absent from the second and third rows of outer hair cells. Ganglion cells were also present although abnormal in appearance. In older homozygous mutants there was a loss of hair cells and spiral ganglion cells. Remaining ganglion cells in this group contained very few cytoplasmic organelles apart from a few hypertrophied mitochondria. In the anteroventral cochlear nucleus, spherical cell soma size was smaller in all homozygous (dfw) mutants than in heterozygous mice and controls. The ultrastructural appearance of the end bulbs of Held in homozygous mutants was abnormal compared with controls, and in the younger group were seen to be swollen, with less distinct synaptic densities and containing large numbers of small synaptic vesicles arranged in clumps. In the older group these synapses were distorted and contained hypertrophied mitochondria and no synaptic densities could be seen, suggesting that these synapses may be non-functional. This study has shown that in homozygous (dfw) mice structural abnormalities occurred not only in cochlear hair cells but also in the spiral ganglion neurones and spherical cells in the cochlear nucleus. It seems likely that these changes are the result of the Pmca2 mutation and the subsequent accumulation of toxic levels of calcium that may lead to alterations in their functional integrity.

Deafferentation-induced changes in protein kinase C expression in the rat cochlear nucleus.

Isoforms of the signal transducing molecule, protein kinase C (PKC), may play a role in neural plasticity following sensory deafferentation. To explore the role of PKC in central auditory plasticity, we studied the effect of auditory deafferentation on the expression of PKC betaI, betaII, gamma, and delta in the rat dorsal (DCN) and ventral cochlear nucleus (VCN), using immunocytochemistry. Male rats were treated with kanamycin and furosemide to induce hair cell loss. At various intervals post-treatment, brains were perfusion-fixed and processed for immunocytochemistry. Following deafferentation, we observed a gradual increase in PKC betaI immunoreactivity (ir) in the deepest layers of the DCN, possibly representing synapses of primary afferents or parallel fibers on unlabeled neurons. Correlated with this, we observed an increase in the number of neurons in the deep DCN that showed PKC delta ir. In controls, we observed PKC gamma ir in small ovoid cells concentrated in the middle layer of the DCN. From days 4 through 14 after deafferentation, we found an increase in the intensity of staining of these cells, with a return toward control levels by day 28. Finally, Purkinje-like cells (PLC) in the VCN, which express only PKC delta in control rats, began to express PKC gamma after deafferentation, correlated with increased expression of calbindin D28k in PLC. Thus PKC isoforms are differentially regulated in the CN following deafferentation, supporting a role for PKC in auditory plasticity.

Age-related reductions in the activities of antioxidant enzymes in the rat inferior colliculus.

The inferior colliculus (IC) is a major relay and processing center of auditory signals in the midbrain and receives inputs from most other auditory nuclei. A number of studies have indicated age-related declines in the GABAergic and excitatory amino acid systems in the IC, including losses in both GABA immunoreactive (+) and GABA immunonegative (-) synapses. The goal of this project was to identify potential biochemical and morphological changes in the IC that may contribute to deficits in the functions of these neurotransmitters, using three age groups of Fischer-344 rats. Homogenates obtained from the IC showed age-dependent reductions in activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), with a concomitant increase in lipid peroxidation. Dephosphorylation of IC homogenates with alkaline phosphatase reduced the activities of SOD and CAT in all age groups, which could be restored by protein kinase C (PKC)-dependent phosphorylation. Restoration of enzyme activity was specific to the PKC-alpha isozyme, but not to the beta1, beta2, delta or gamma forms. No age-dependent change in the levels of PKC isoforms (alpha, beta1, beta2 and gamma) was detectable in IC homogenates. Morphological analyses indicate decreases in mitochondrial density in the somata of both GABA+ and GABA- IC neurons in 19- and 28-month-old rats when compared to 3-month-olds, along with significantly higher matricial abnormalities. These data indicate age-related increases in oxidative stress in the IC, which could be partially restored by PKC. The progressive increase in oxidative stress with age may underlie changes in neuronal morphology and function of the IC.