Katherine J. Rennie - US grants

DESCRIPTION: The study proposes to use patch-clamp techniques to study the modulation of K+ conductance in Type I vestibular hair cells. The investigato hypothesize that alterations in extracellular K+ and phosphorylation of an intracellular enzyme could lead to alterations in K+ conductance in response t a depolarizing stimulus. The second aim of the study is to record ionic currents in utricular cells in situ, and contrast these recordings with those obtained from isolated hair cells.

DESCRIPTION: Experiments described in this proposal will address the hypothesis that the membrane properties of type I Vestibular hair cells can be modulated and that one source of this modulation is through transmitter release from the apical regions of the calyx terminals which innervate type I cells. Type I hair cells, found only in amniotes, are the only hair cells 'innervated by an afferent calyx that encases the basal pole of the cell. Synaptic transmission between type I cell and its associated calyceal ending is believed to be chemically mediated. Depolarization of the type I hair cell, through deflection of the hair bundle, is assumed to increase the amount of an unidentified neurotransmitter, most likely glutamate or GABA, released from the hair cell. Although the calyx fiber is known to encode afferent information from the type I hair cell, morphological evidence suggests that the upper regions of the calyx may release neuroactive substances onto the hair cell. In addition, Vestibular efferent fibers make synaptic contact with the outer face of the calyceal membrane. Thus the type I hair cell is postulated to be a presynaptic sensory cell under local control from the calyx and also possibly under central control via action of the efferents on the calyx. Recordings from solitary type I cells indicate that the whole cell ionic currents are large and variable in magnitude. It is hypothesized that these currents may be physiologically modulated, an obvious source of modulation would be through transmitter release from the calyx. Modulation of type I hair cell conductances would alter the hair cell's response to transducer current which could confer considerable flexibility on the processing of mechanoelectrical signals by the type I hair cell. To test the hypothesis that type I currents can be modulated, experiments will be carried out on dissociated gerbil type I cells. The identity of receptors on the calyceal membrane will be established by application of putative afferent and efferent transmitters (and their antagonists and agonists) to isolated calyces in whole cell patch-clamp or to outside out patches. Recordings from type I cells in situ will establish whether ionic currents in type I cells contacted by calyces are similar to those in isolated cells and the effects of nerve simulation on type I cell responses will be assessed, to verify whether type I cell conductances are under feedback control. Results from these experiments will provide information on the synaptic physiology of mammalian type I hair cells and their calyces.

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to understand synaptic transmission at the vestibular type I hair cell/calyx synapse. Three classes of morphological afferents have been described in the amniote crista and utricle. Calyx units contact type I hair cells (HCI) exclusively, bouton units contact type II hair cells only & dimorphic units receive innervation from both bouton & calyx fibers (Goldberg, 2000). The physiological response dynamics of these three classes of fibers vary, with calyx units having the most irregular firing pattern & the lowest gains to rotational stimuli (Baird et al. 1988; Lysakowski et al. 1995). The reasons for these variations are unclear, but are hypothesized to include differences in hair cell mechano-electrical transduction (MET) properties & differences in the biophysical membrane properties of primary vestibular afferents. We will study HCI & associated calyx afferents to determine how firing patterns in this unique terminal are shaped by both pre- & post-synaptic mechanisms. In Aim 1, patch clamp techniques will be used to study ionic conductances & transmitter release in a newly developed preparation of calyx terminals isolated together with HCI from gerbil vestibular organs (Rennie & Streeter, 2006). Excitatory postsynaptic currents (EPSCs) resulting from hair cell transmitter release will be recorded from calyx terminals under a variety of conditions. In Aim 2 we will record MET currents & receptor potentials from HCI during displacement of the hair bundle with a stiff probe in a wholemount utricle preparation. In Aim 3, mathematical modeling techniques will be employed to simulate HCI & calyx responses to glutamate. The passive electrical properties of the calyx & attached axon will be simulated with a segmented computational model in the NEURON programming environment. Na+, Ca2+ and K+ channels will be modeled with Hodgkin-Huxley style rate constants using the experimental data obtained in Aims 1& 2. A genetic algorithm wiil be used to optimize the kinetic parameters for the activation & inactivation of ionic conductances. Dizziness is one of the most common medical complaints. Understanding the basic cellular mechanisms of balance sensation is essential to lay the groundwork for identifying causes & cures for this debilitating condition. The combination of experimental & modeling approaches will elucidate how sensory information is transformed by HC1 & converted into a neural code by their afferents. [unreadable] [unreadable] [unreadable]

Project Summary Approximately 8 million adults in the US suffer from balance impairment due to damage to the peripheral vestibular system, but effective treatments for balance dysfunction are lacking. Vestibular hair cells within vestibular canal and otolith organs convert motion into receptor potentials and sensory information is relayed to the brain by action potentials in vestibular afferent nerves. Afferents in central zones (CZ) of vestibular neuroepithelia exhibit different responses to vestibular stimuli than afferents in peripheral zones (PZ). The nature of the neural code conveying vestibular information in distinct afferent types is poorly understood. There are 3 types of vestibular afferents: calyx-only afferents innervate one or more type I hair cells, bouton dendrites innervate type II hair cells and dimorphic afferents contact both hair cell types. Our goal is to elucidate distinct action potential firing mechanisms in afferents with calyx terminals to better understand vestibular coding. Calyx-only afferents are present solely in CZ and have irregular firing patterns, whereas dimorphic afferents exist in both CZ and PZ and have regular firing patterns. To achieve our goal we will refine novel preparations of vestibular cristae and utricles, developed by our laboratory, as tools to study calyx-bearing afferents in CZ and PZ of rodent neuroepithelia. We will employ electrophysiological, hair bundle stimulation, immuno- histochemical and pharmacological approaches to characterize ion channels in CZ and PZ afferent fibers in developing and mature epithelia. In Aim 1 we will determine the contributions of K+ channels to action potential firing in CZ and PZ afferents. Aim 2 will test the hypotheses that tetrodotoxin (TTX)-sensitive Nav1.6 Na+ channels contribute uniquely to action potential firing in mature PZ dimorphs, and that TTX-insensitive Na+ channels are transiently expressed during development. In Aim 3 we will incorporate ion channel data from Aims 1 and 2 into a novel, custom-written three dimensional mathematical model of the calyx to provide insight into our zonally-driven experimental findings.To determine how channel localization directly impacts action potential firing, identified channel types will be strategically placed on the inner and outer faces of the calyx terminal and associated axon and channel density varied. Our results will clarify how sensory information is conveyed and how zonal encoding is generated within segregated vestibular afferents. Our data will inform development of vestibular neurotherapeutics targeting specific groups of ion channels in afferent nerves. Existing vestibular prosthetic implants attempt to restore normal vestibular function by direct electrical stimulation of vestibular afferents, but implants that restore function to both otolith and semicircular canal afferent neurons do not yet exist. Our results will provide important new information on vestibular afferent coding that could inform development of and drive new paradigms in vestibular implants.

Project Summary Vestibular dysfunction becomes more prevalent with age and it is estmated that more than 80% of people over 80 years old experience dysfunction5. Furthermore, approximately 8 million adults in the US suffer from balance impairment due to damage to the peripheral vestibular system, but effective treatments for balance dysfunction are virtually non-existent. Vestibular hair cells within vestibular canal and otolith organs convert hair bundle motion into receptor potentials and sensory information is relayed to the brain by action potentials in vestibular afferent nerves. Afferents in central zones of vestibular neuroepithelia exhibit different responses to vestibular stimuli than afferents in peripheral zones. There are three types of vestibular afferents: calyx-only afferents innervate one or more type I hair cells, bouton dendrites innervate type II hair cells and dimorphic afferents contact both hair cell types. Calyx-only afferents are present only in central zones and have irregular firing patterns, whereas dimorphic afferents exist in both zones and have regular firing patterns. We will study age-related dysfunction in calyx-bearing afferents in gerbil vestibular organs using novel preparations developed in the laboratories of the principal investigators. We will use electrophysiological, hair bundle stimulation, immunohistochemical and behavioral approaches to address age-related changes in mature and aged vestibular epithelia. In Aim 1 we will determine if functional changes in vestibular hair cell mechanotransduction and/or basolateral currents occur with age. Aim 2 will test the hypotheses that synaptic degeneration of calyx terminals will manifest as morphological uncoupling of type I hair cells from their associated calyces and deficits in vestibular evoked responses and behaviors. In Aim 3 we will directly investigate changes at the type I hair cell/calyx synapse by recording spontaneous activity and responses to hair bundle stimulation in mature and aged calyx afferents. Our investigative team is uniquely positioned to carry out the proposed studies. Results from this work will provide new information on how the aging process impacts peripheral vestibular signals and may inform development of vestibular neurotherapeutics targeting afferent nerves in order to restore normal vestibular function.