Maria E. Rubio - US grants

Our long-term goal is to understand the role of hearing and deafness in the distribution and expression of transmitter receptors. In this proposal, we will focus on the auditory nerve-cochlear nucleus (CN) interface with a special focus on the synaptic circuitry of three principal projection neurons: the fusiform cell (FC) in the dorsal cochlear nucleus, and globular and spherical bushy cells (GBC and SBC) in the anteroventral cochlear nucleus. These cells play key roles in the integration of converging synaptic inputs from diverse sources. They form part of the neuron population that initiates the ascending auditory pathways by which auditory information is communicated to higher centers. In this proposal, we have two specific objectives. In Aim 1, we will determine whether and to what extent hearing loss leads to changes on the expressionand subunit composition of glutamate receptors at the postsynaptic membrane of FC, GBC and SBC opposed to auditory nerve synapses. This study will use carboplatin-induced sensorineural deafness to test the hypothesis that the molecular composition of the glutamate synapse will change after deafening. We will use immunogold labeling and electron microscopy to examine the effects of deafness on the distribution and type of receptor subunits on these cells as compared to that found in hearing littermates. In Aim 2. we will determine whether conductive hearing loss leads to changes in the expression and subunit composition of glutamate, glycine and GABAA receptors at the postsynaptic membrane of FC, GBC and SBC. We will test the hypothesis that a reduction in acoustic stimulation leads to similar types of receptor remodeling. Using ear plugs, we will attempt to determine the effects of "hearing reduction" on the expression of glutamate, glycine and GABA receptors on these CN neurons. The proposed research combines 3-D reconstruction and morphometric analysis together with quantitative immunocytochemistry at the light and electron microscopy level. Through these studies we will determine the immediate morphological and molecular changes caused by sensorineural and conductive hearing loss on the main neurons in the cochlear nucleus. The results of these studies may reveal the nature of molecular change induced by deafness and loss of auditory nerve activity. They will have direct relevance to strategies that attempt to preserve or replace hearing (via cochlear implants) in cases of congenital deafness, and may lead to treatment paradigms for tinnitus. The proposed research will make novel contributions to the field of glutamatergic brain plasticity and auditory neurobiology.

DESCRIPTION (provided by applicant): The auditory nerve (AN) transmits all auditory information from the cochlea to the brain. In the cochlear nucleus (CN), AN fibers bifurcate to innervate multiple cell populations, including bushy cells (BCs) in the ventral CN, and fusiform cells (FCs) in the dorsal CN. These two cell types differ significantly in their ability to encode temporal properties of sound stimuli. BCs project to binaural circuits in the superior olivary complex and encode spectral and temporal characteristics that allow sounds to be localized in the horizontal plane. FCs project to monaural circuits in the inferior colliculus and detect spectrl cues for localizing sounds in the vertical plane. AN synapses on BCs and FCs are both glutamatergic and involve AMPARs as major postsynaptic glutamate receptors. At AN-BC synapses, synaptic transmission is extremely fast and reliable to preserve information contained in the timing of AN spikes. At AN-FC synapses, synaptic transmission is significantly slower than at AN-BC synapses. Understanding the synaptic mechanisms that make AN-BC synapses faster than AN-FC synapses has been an important question that has been intensely studied. However, which specific AMPAR subunits actually mediate fast synaptic transmission at AN synapses is still unresolved. The goal of the proposed studies is to provide understanding of the functional role of GluA3 AMPAR subunits at AN synapses on brainstem neurons and the sensitivity of AN synapses to auditory experience. Data obtained from this proposal will advance understanding of the cellular mechanisms underlying the temporal precision of sound coding in the normal and in the hearing impaired. Thus in Aim 1 we will test the hypothesis that GluA3 in AN-BC synapses is the AMPAR subunit that determines fast AMPAR kinetics. Aim 2 will test the hypothesis that increase in expression and localization within the PSD of GluA3 AMPAR subunits mediates the experience-dependent plasticity of AN-BC and AN-FC synapses. To achieve these goals, we will combine hearing tests (auditory brainstem responses, ABRs) to analyze the ability of the brainstem to respond to sound stimuli in vivo, quantitative ultrastructural and molecular techniques, genetic approaches (knockouts) and electrophysiology in acute brainstem slices of adult normal hearing and monaurally earplugged mice. Specifically, we will use freeze-fracture and postembedding immunogold labeling, qRT-PCR together with whole-cell recording to identify morphological, molecular and functional alterations at AN synapses. The results of our studies can be applied to efforts to optimize strategies for treating hearing loss and other hearing disorders. A large body of evidence indicates that the auditory system is highly specialized. Systematic, rigorous studies of the synaptic mechanisms underlying the specializations will both suggest and inform rational therapeutic approaches.

Project Summary All information about the acoustic environment is carried from the inner ear to the CNS by the afferent fibers of the cochlear nerve. Rapidly gating AMPA glutamate receptors (AMPAR; GluA2, GluA3 and GluA4 subunits) mediate synaptic transmission at the mature synapse between the inner hair cells (IHC) and the afferent fibers of the cochlear nerve (IHC synapse). However, the contribution of each type of AMPAR subunit to overall glutamatergic receptor function and afferent transmission/sensitivity in the cochlea is poorly understood. Understanding this process is important because glutamate excitotoxicity through AMPAR has been implicated in the pathogenesis of hearing loss caused by noise, ischemia and aging. Sex differences in the vulnerability to hearing loss occur in humans. We therefore began investigating the contribution of AMPAR subunits to transmission at the IHC synapse and whether there are sex-specific differences in AMPAR subunits that contribute to sound-induced cochlear damage and hearing loss. Based on functional and ultrastructural preliminary data, we now hypothesize that ?GluA3 AMPAR subunits have a critical role in the sexually dimorphic vulnerability to hearing loss?. To define mechanistically how GluA3 contributes to the structural and molecular components of IHC synapses and to sex differences that underlie the hypersensitivity to sound- induced cochlear damage, we will use a powerful combination of functional (ABRs, DPOAEs), immunocytochemical (confocal microscopy), biochemical, qRT-PCR, and ultrastructural approaches to test the following hypotheses. In Aim 1, we will determine whether GluA3 promotes the abundance of GluA2 at IHC synapses. In Aim 2, we will determine whether GluA3 at IHC synapses protects mice from sound-induced cochlear damage. Aim 3, based on published data and our preliminary findings, we propose the hypothesis that in the absence of GluA3, ovarian hormones facilitate the hypersensitivity to sound-induced cochlear damage, while androgens have protective effects. These proposed studies are the first to address the important question of how changes in AMPAR subunit composition lead to sex differences in hearing loss.