Jonathan J. Art, Ph.D. - US grants

The goal of the proposed research is to characterize the cellular mechanisms that contribute to tuning in turtle cochlear hair cells. By understanding how these cellular mechanisms contribute to frequency selectivity, we will be able to assess their limitations and applicability to hearing in higher vertebrates including man. A combination of whole-cell and single-channel patch-clamp techniques will be used on solitary cells isolated from identified regions of the turtle cochlea. The first aim is to analyze the mechanical response of the hair-cell ciliary bundle, and to determine the role of this response in the overall transduction mechanism. The source, strength and voltage-sensitivity of the ciliary motion will be determined. The ability of the transducer channel to detect this motion will be ascertained, and any variation in the size and voltage-sensitivity with characteristic frequency will be assessed. The second aim is to characterize further the conductances in the basolateral hair-cell membrane implicated in electrical resonance. Changes in the size and kinetics of the membrane currents in cells of known characteristic frequency will be analyzed using intracellular perfusion. Specific protocols address the isolation inward currents due to the apical transducer and the basolateral calcium current, the characterization of possible electrogenic transport of calcium out of the cell, and a separation of the calcium from the calcium-activated potassium currents in a single cell. The third aim is to analyze the kinetic behavior of the calcium-activated potassium channel and to characterize the calcium- and voltage- sensitivity. Since the kinetic behavior of this current is highly correlated with the characteristic frequency to which a hair cell is tuned, it is presumed to play a major role in frequency selectivity. Simultaneous cell-attached single-channel and whole- cell recording will be used to compare the behavior of the single channel and the macroscopic calcium-activated potassium current. The proposed research will analyze these ionic currents to assess whether hair-cell tuning due to basolateral ionic conductances coexists with, or is enhanced by, mechanisms acting via the transduction process in the ciliary bundle. The ionic currents measured under voltage-clamp will be used to reconstruct the membrane resonance seen in current-clamp.

DESCRIPTION (provided by applicant): A multiphoton laser microscopy (MPLM) system from Prairie Technologies is requested. The custom system will be assembled in collaboration with the Northwestern University Multiphoton Center Core. The system, developed by Prairie and Northwestern Scientists with software and hardware for simultaneous physiological measurements, consists of two stations: one capable of imaging tissue of live animals, the other for tissue slices. The instrument will be housed in the Research Resources Center (RRC) of the University of Illinois at Chicago. The rapidly expanding Imaging Facility of the RRC provides all research groups at UIC and surrounding institutions access to a full range of EM, optical and image analysis capabilities. The requested MPLM and associated technologies are uniquely suited to imaging of molecules and structures in cells located deep within live tissue sections or living animals. Experiments that require these methodologies cannot be undertaken with the instruments currently at UIC. The intellectual and technological resources necessary to understand complex pathological processes that involve intra and intercellular transport of molecules, development in various model systems, cytoskeletal remodeling, axonal transport, immunology, viral entry, tumor formation, signaling and molecular imaging are in place. The MPLM technology will be critical in advancing our research programs as well as fostering interactions among investigators with diverse but compatible interests. Our overall objective as a group is to understand the dynamic molecular events that occur within the context of specific tissues. This is particularly critical in complex tissues like the nervous system, developing embryos, endocrine organs, where relationships between different cell types cannot be replicated in isolated cultures. MPLM imaging will provide us with heretofore unavailable windows on basic molecular interactions among key proteins involved in intracellular trafficking, cell signaling, and pathogenesis. Our strengths, as an interdisciplinary research group with basic and applied research programs in Development, Cell Physiology, Neuroscience, Virology and Immunology, provide the basis of the need for this instrumentation. The addition of the proposed MPLM system will provide a unique capability relevant to several key NIH funded investigators where we have a nationally-recognized track-record in axonal transport and neurodegeneration, virus entry and trafficking, cytoskeletal protein remodeling, cancer, immunology, signal transduction, and molecular imaging. We believe that the insights gained will have the potential for pioneering new therapeutic targets and strategies in HIV prevention, cancer, autoimmune disease, diabetes, and neurodegeneration.

Project Summary/Abstract In the vestibular system, accelerations of the head are transduced and processed prior to transmission to the brain. Prior research has shown that responses of primary afferent neurons from the semicircular canal deviate from canal biomechanics, and has implicated signal processing by the hair cell?primary afferent synapse in shaping the response. Afferent processing is complex due to both the convergence of multiple hair cells onto a single afferent, and three modes of synaptic transmission between hair cell and afferent. Type I hair cells are enveloped by their afferent in a calyceal ending that forms a restricted volume, or cleft, where rapid excitatory synaptic transmission, via glutamatergic AMPA receptors, is modulated both pre- and post-synaptically by K+, H+, and Ca2+ accumulation. By contrast, type II hair cells make synaptic contact either onto conventional bouton endings, or onto the external face of calyces that envelop type I hair cells. The afferent taxonomy is equally complex, and includes three major classes of hair cell to afferent convergence. The simplest are the pure bouton afferents, where hair cells converge onto their afferent via bouton endings. Increased complexity is found at calyceal endings, composed of either a simple calyx in which the afferent envelops a single hair cell, or complex calyces, where the afferent encompasses two or more. The most complex afferents are the dimorphic endings that contact both type I and type II hair cells via a combination of bouton and inner? and outer?face calyceal synapses. Attempts to correlate and rationalize afferent physiology with morphology have been impeded by two outstanding problems. The first is that morphophysiological studies allow a correlation between the number of bouton and calyceal endings and afferent physiology, but cannot estimate the number of type II hair cells that synapse onto the external face of a calyx. As a consequence, the external face synapses have remained cryptic in prior studies, and the impact of these contacts on afferent physiology remain obscure. The second problem is that little is known about the calculus of synaptic transmission at any ending, or how the geometry of quantal and non-quantal transmission determines the dynamics of afferent discharge. To surmount these problems and to more fully understand the variations in synaptic transmission, we will capitalize on our recently developed simultaneous recording from hair cells and their associated afferents to characterize synaptic transmission between type I and type II hair cells in dimorphic endings. Specifically, we will characterize the rapid glutamate?mediated transmission and its modulation by ion accumulation in the cleft between the type I hair cell and the inner face of the afferent calyx. We will contrast this mode of transmission to that between the calyx and type II hair cells that synapse onto the outer face of the calyx. Finally, we will characterize the type II hair cell synaptic input onto boutons, as well as the electrical properties of the dendritic arbor connecting type II hair cell boutons with the parent fiber in dimorphic afferents.

Project Summary Prior research demonstrates that afferent responses from semicircular canal cristae and otolith organ maculae deviate from the coherent mechanical stimulation imparted by their overlying accessory structures. This implicates further processing by hair cells (HCs), primary afferents, and the HC - afferent synapse. Processing is complicated by the parallel modes of synaptic transmission between HCs and afferents, and the convergence of multiple HCs onto a single afferent. In the vestibular periphery progress towards describing variations in afferent discharge in terms of the time course of underlying voltage- and ion-sensitive conductances has been impeded by two major anatomical features of the vestibular epithelia: 1) access to HCs and afferents in their native bi-ionic (endolymph - perilymph) environment is mechanically impeded, so there are few in situ recordings to serve as controls for pathophysiology in recordings made from isolated cells or epithelial explants; and 2) analysis of integration at the level of a ramifying afferent is complicated by multiple HC convergence onto each, and the impossibility of using a single patch-electrode to space-clamp a distributed afferent arbor. To overcome the inaccessibility problem associated with obtaining physiological data for vestibular HCs in their native environment, we will measure voltages using slow, potentiometric (Nernstian) dyes, whose equilibrium partition (concentration) is voltage-dependent. These dyes will be superfused across the vestibular and auditory epithelia in a turtle half-head preparation. The voltage-dependent fluorescence of slow redistributive dyes will be measured using multiphoton microscopy (MPM), and calibrated using microelectrode recordings from HCs, afferents and supporting cells via the readily accessible perilymphatic space of the auditory papilla. The type I HC/calyceal afferent synapse is relatively compact electrically, but HC convergence onto a single afferent over distances of 10s to 100s of microns makes it difficult to address the passive and active properties of an afferent arbor. As a consequence, it remains problematic to characterize afferent integration using conventional electrophysiological techniques. We will examine afferent convergence by patch recording single afferents using electrodes filled with electrochromic voltage-sensitive dyes (VSDs). Steady-state depolarizations and hyperpolarizations of the afferent via the patch electrode will be used to optically characterize the passive cable properties of the ramifying afferent using lattice light sheet microscopy (LLSM). Pulses of current injected through the patch electrode, or electrical stimulation of the nerve - phase-locked to image acquisition on the LLSM - will be used to image and average orthodromic and antidromic AP propagation in the parent axon and throughout the afferent arbor. We propose novel approaches to make highly significant measures that are currently unavailable. By using optical and electrode recordings to characterize the cellular potentials and the afferent convergence, these experiments have the potential to make large and durable contributions to the field.