Catherine Jeanne Chalenski Weisz - US grants

DESCRIPTION (provided by applicant): The cochlea contains two types of primary afferent neurons, the Type I and Type II spiral ganglion neurons. 95% of the spiral ganglion population consists of the Type I neurons, which encode acoustic stimuli. The remaining 5% of the spiral ganglion population, the Type II neurons, ramify extensively among outer hair cells (OHCs). Little is known about their electrical properties as only a few recordings have been performed from the neurons. This lack of functional information precludes hypotheses as to the role of the Type II afferents in perception of auditory stimuli. We have developed a technique that allows gigaohm-seal intracellular recordings in Type II afferent dendrites near their synaptic inputs. Use of a neuronal tracer allows identification of the neurons based upon comparison to the known dendritic morphology well described in the literature. We propose to demonstrate that synaptic inputs to the Type II afferents from OHCs are functional, and to describe the strength of these inputs to Type II neurons. Comparison of the activation induced by synaptic inputs to the threshold for fiber activation as determined within the aims of this proposal will allow estimation of the level of stimulation of OHCs that may be necessary to induce supra-threshold activation of the Type II afferent. Electrophysiological parameters of the Type II dendrite will be incorporated into a cable model of the neuron. This can be used to determine the likelihood that stimuli will produce enough activation of the dendritic field to generate action potentials that may then be transmitted centrally to be incorporated into our perception of auditory stimuli. Purines stimulate the Type II afferents, both directly and by eliciting synaptic input from OHCs (preliminary data). While ATP serves many purposes, it can also be released from damaged cells and may be involved in signaling of cell damage in response to loud sounds. Other compounds implicated in cellular damage will be tested for their ability to activate Type II dendrites to further probe this hypothesis. Further, we will begin to describe processes by which the neurons are sensitized. Activation of UTP, an agonist at metabotropic P2Y receptors, stimulates Type II dendrites. Therefore, we will use pharmacological tools to manipulate second messenger pathways that may be downstream of P2Y activation in order to determine their role in Type II dendrite excitability. We will also test other compounds that may similarly initiate these second messenger cascades, such as Substance P, for their actions on activation and sensitization of Type II afferent dendrites. Prior to preliminary data presented for this proposal, nearly nothing was known regarding the activity in response to stimuli of one of the only two classes of primary auditory neurons. Therefore, analysis of the functions of Type II afferent neurons is crucial to a broader understanding of perception of sound. PUBLIC HEALTH RELEVANCE: The cochlea contains two types of neurons that transmit sensory information to the central nervous system, the Type I neurons which carry the acoustic information of sound timing, intensity and frequency, and the poorly understood Type II neurons about which little is known. This research proposal aims to develop techniques to measure electrical activity within the Type II neurons, which may enable a determination of their functional roles. An understanding of responses of both classes of auditory neurons is necessary for a complete understanding of perception of sound.

DESCRIPTION (provided by applicant): The neurons of the lateral superior olive (LSO) in the auditory brainstem receive sound information from both ears. Inputs from the ipsilateral ear are via the cochlear nucleus, which gives rise to an excitatory, glutamatergic projection to the LSO. Inputs from the contralateral ear project to the contralateral cochlear nucleus, then through the medial nucleus of the trapezoid body (MNTB), a group of neurons that convert the glutamatergic signal to an inhibitory, glycinergic projection. The combination of inputs from the two ears determines the ability of LSO neurons to compute the location of sound in space around the head. Early in development the MNTB neurons also release the neurotransmitters GABA and glutamate. The purpose of these neurotransmitters in the proper structural development of neurons from the two ears is not very well understood. If glutamate is absent from the MNTB neurons, it has been shown that there is an improper refinement of patterns of neurons from the two ears that encode the same frequency onto a single LSO neuron. Less is known about the function of GABA in these synapses, but it has been shown that GABA release can cause a calcium rise in the LSO neurons, which may be important for formation of synapses between the neurons. This proposal aims to study the role of GABA in development of the projections from the MNTB to the LSO. Experiments will be performed in brain slices from mice containing both MNTB neurons and their projections to LSO neurons. Techniques will include whole-cell electrophysiology and 2-photon imaging to measure the patterns of activity in neurons when GABA signaling is altered pharmacologically. Tools including fluorescent dyes that measure changes in calcium signaling (indicative of cellular activity) and 'optogenetic' tools that allow stimulation of individual neurons using laser light will be used. The proposal also includes electron microscopy experiments to investigate the subcellular structures at the synapses between MNTB and LSO neurons in order to measure how GABA receptors are involved in the proper formation of synapses between the MNTB and LSO. This research will also have implications for investigation of GABA signaling in other brain regions. Research into the proper formation of neuronal circuits that encode sound localization is crucial for a greater understanding of hearing, both in animals and in humans. Understanding how neurons properly connect in developmental stages guides understanding of what can go wrong in situations in which normal hearing is compromised through aberrant development or through injury. A full understanding of the complexity of the perception of sound will guide refined therapies for treatment of hearing disorders.

This is a new laboratory. The focus this initial year has been in areas of personnel, scientific training, modifications to lab facilities, procurement of scientific equipment, establishment of animal protocols and mutant mouse colonies, and generation of preliminary data. Specifically, a lab manager and post-baccalaureate researcher were hired. Two post-doctoral research fellows were recruited, and will join the lab in fiscal year 2017. Minor modifications and repairs were made to the lab space, primarily in chemical exhaust systems. Lab supplies and small equipment were purchased and the lab has been set up. Finally, major equipment for electrophysiological recordings and imaging of activity in single neurons has been installed and tested, lab staff are being trained to conduct experiments, and preliminary data has recently been collected. Synaptic inputs of olivocochlear neurons in the brainstem and synaptic outputs in the cochlea Medial and lateral olivocochlear (OC) neurons have cell bodies in the brainstem, where they receive synaptic inputs conveying sound information from the cochlea via the cochlear nucleus. The activation and inhibition of OC neurons by this direct pathway from the ear is at early stages of investigation at the synaptic level. The neurons project axons to the cochlea, where they innervate hair cells and spiral ganglion neurons that comprise the first synapse in the ascending auditory system. Great strides have been made by other laboratories in determining the behavioral effects of the MOC neurons in the cochlea. MOC neurons decrease the mechanical movement of the basilar membrane by inhibiting cochlear outer hair cells (OHC). This effect is implicate in improved hearing in noise, protection of the cochlea against sound trauma. Little is known about the synaptic outputs of LOC neurons in the cochlea, but they may act to either increase, or decrease, auditory nerve activity depending on the specific neurons or synapses activated, and the neurotransmitters employed. We aim to understand the diversity of both synaptic inputs and outputs of OC neurons, in order to fully understand how the neurons are activated, modulated, and how their properties may change in pathological situations such as in tinnitus, hyperacusis, or acquired deafness. Experimental accomplishments include development of a computational model of MOC neurons, including addition of intrinsic electrical conductances from experimental data obtained from published work from other labs, and comparison of this computational model to recently collected experimental data from whole cell patch-clamp electrophysiological recordings of identified MOC neurons in our own laboratory. Two lab members were recently trained in this electrophysiological technique, and are generating preliminary data. In these brainstem slice experiments we also are beginning a detailed investigation of synaptic inputs to MOC neurons. These experiments will elucidate the mechanisms of synaptic activation of MOC neurons, which in turn drives their inhibition of mechanical activity in the cochlea, thus shaping the cochlear response to sound. In parallel, equipment to allow dendritic electrophysiological recordings from spiral ganglion afferent neurons is being set up in the laboratory. Behavioral assessment of olivocochlear function The medial olivocochlear system, a component of the final stage of the descending auditory system, is implicated in diverse effects on hearing including detection of salient sounds such as speech in noise, protection of the cochlea against noise-induced trauma, and may have altered activity in pathological conditions such as tinnitus and hyperacusis. Recent reports from other laboratories provide contradictory evidence regarding the specific neurons that activate the MOC reflex. In a collaborative project with Dr. Rebecca Seal at the University of Pittsburgh, we have developed a mouse line that will provide more precise control of afferent signaling through type II spiral ganglion afferent neurons. We are in the planning stages of a collaborative project with Dr. Tracy Fitzgerald, Director of the NIDCD Animal Auditory Testing Core Facility, to test the function of the MOC system in control and in these mutant animals. These experiments will elucidate the pathways through which the MOC reflex is activated, distinguishing between inputs from type I vs type II spiral ganglion afferents. The experiments will employ measurements of distortion product optoacoustic emissions (DPOAEs), a test of OHC function. The change in DPOAE strength by contralateral suppression, a measure of MOC activity, will be used to assess the MOC function in control and mutant mice. These experiments will determine whether type II spiral ganglion afferent neurons indeed contribute to the MOC reflex. A review of recent literature assessing the role of MOC neuron function in humans with tinnitus and hyperacusis resulted in a journal club style publication, reference #1. VGLUTs in OHCs, and the central innervation patterns of type II cochlear afferent neurons In a collaborative project with Dr. Rebecca Seal at the University of Pittsburgh, the specific vesicular glutamate transporters (VGLUTs) employed by OHCs to load glutamate into presynaptic vesicles for release onto spiral ganglion afferents was determined. This led to generation of mutant mice lacking the VGLUTs from either inner hair cells (IHC) or OHC, or both. Use of these mutant mice allows isolation of afferent signaling by either type of hair cell, in order to determine the unique contribution that each pathway makes to perception of sound. We used these different mouse lines in an experiment in which the animals were exposed to noise, then their cochlear nuclei assessed for activation of the immediate early gene c-Fos. We determined that the OHC-type II afferent pathway can indeed respond to acoustic signals, and evoke activation of neurons in the brainstem. A paper describing this work is currently in preparation. Glutamate receptors in type II spiral ganglion afferent neurons Cochlear OHC were recently found to release glutamate onto type II spiral ganglion afferent neurons. However, the post-synaptic glutamate receptors present on the type II afferent neurons remained unknown. In a collaborative project with Dr. Paul Fuchs and Dr. Elisabeth Glowatzki, it was determined using immunohistochemistry and electrophysiology techniques that type II spiral ganglion neurons contain GLUA2 AMPA receptors, and that the receptors are still present in mature animals. This project resulted in a publication, see reference #2. GABAergic spillover between MNTB presynaptic terminals in a sound localization circuit This is a completed project performed with Dr. Karl Kandler at the University of Pittsburgh. Neurons of the lateral superior olive compare sound intensity differences at the two ears to determine the location of a sound in space. They receive excitatory, glutamatergic inputs from the ipsilateral ear, and inhibitory, glycinergic inputs, from the contralateral ear via neurons of the medial nucleus of the trapezoid body (MNTB). In development, neurons of the MNTB also release glutamate and GABA onto LSO neurons, but the role of this neurotransmitter co-release is poorly understood. We used electrophysiological recordings from LSO and MNTB neurons, paired with live cell imaging, neurotransmitter uncaging, and electron microscopy techniques, to show that GABA released from MNTB neurons spills over to neighboring neurons. It then acts at pre-synaptic GABA A receptors to stimulate the presynaptic terminals, evoking excitation that can induce additional neurotransmitter release at a slight delay. This project resulted in a publication, see reference # 3.