William Michael King - US grants

Our ability to visually identify small objects or to read while we walk, run, or ride depends critically on the vestibulo-ocular reflex (VOR) to minimize image motion across the retina. The basic organization of this reflex appears deceptively simple, a three neuron arc that processes sensory signals to produce eye movements (E + H = O). We now know, however, that the 3 neuron arc is but one of the many pathways used to produce the VOR, and that other, less direct, pathways process essential signals for the reflex. At least 4 nuclear groups, identified by their direct connections with vertical oculomotoneurons, are involved in the vertical VOR: the vestibular nuclei, the vestibular y-group, the INC, and the rMLF. We know the discharge patterns or signals carried by neurons in these nuclei, and we can make reasonable assumptions about what signal is conveyed to motoneurons by each of these premotor sources. Our long range goal is to analyze how the oculomotor system generates these signals, and how they are combined to produce normal vertical conjugate eye movements, but this proposal is aimed specifically at the contribution of the INC and the vestibular y-group. These nuclei carry signals closely related to slow phase eye velocity and to eye position during the vertical VOR, smooth pursuit, and fixations. The vertical VOR is an integrated response to afferent signals arising from the semicircular canals, the otolith organs, and the visual system. The secondary goal of this proposal is to learn how these afferent signals interact and are combined to produce the vertical VOR. These aims are specifically delineated as research needs and opportunities in conjugate eye movement control in the NIH publication, Vision Research: A National Plan 1983-1987.

Botulinum A toxin (BTX) paralyzes neuromuscular transmission by blocking release of acetylcholine from nerve terminals. In recent years, small quantities of BTX have been injected into extraocular muscle of strabismic humans to induce corrective changes in ocular alignment. For many patients, the corrective changes in ocular alignment persist long after the direct paralytic effects of BTX have worn off. These findings suggest that peripheral or central adaptive processes, triggered by BTX paralysis, induce ocular alignment. Despite extensive clinical experience with BTX, however, the nature of these processes is obscure. This study will employ a multidisciplinary approach designed to identify and study the character of these adaptations. The study will examine infant and adult animal models of strabismus as well as human patients selected for BTX therapy. Five specific experiments are planned: 1) To measure passive orbital stiffness and hysteresis in adult and infant monkeys. Preliminary studies in our laboratory suggest that orbital stiffness changes are sensitive indicators of structural changes in the globe-muscle system. 2) To create a clinically relevant animal model of strabismus by performing myectomies of a horizontal rectus muscle. This model will be used to study the effects of BTX treatment to relieve or prevent muscle contracture. 3) To analyze binocular eye movement patterns in monkeys after myectomy and/or BTX treatment. 4) To study ultrastructure in paired agonist-antagonist extraocular muscles after BTX treatment. 5) To compare and study eye movements of human patients treated with BTX. Taken together, our findings should provide significant insights into central and peripheral adaptive mechanisms relevant to the development of strabismus, and its treatment by BTX therapy.

DESCRIPTION: (Adapted from the applicant's abstract.) The vestibulo-ocular reflex (VOR) is essential for normal visual acuity because it acts to stabilize visual images on the retina against movements of the head and body. Head and body movements are sensed by the semicircular canals (angular accelerometers) and the otolith organs (linear accelerometers). The shortest neural pathway is a 3-neuron arc consisting of primary receptor neurons, interneurons in the vestibular nuclei, and motoneurons that drive the muscles of the eye. The reflex is extremely fast, exhibiting a latency as short as 8 ms in the monkey. In general, if one is viewing a target in the distance, the compensatory eye movements should be equal in magnitude but opposite in direction to the perturbing head movement (Gain - -1). Frequently, however, one views an object in extrapersonal space, e.g., something held in the hands. For near targets such as these, there is no single correct gain for the VOR, since the axis of head rotation cannot be coincident with the rotational axes of the eyes. Normally the center of rotation is behind the eyes (e.g., above the spinal cord for left or right head turns), and geometric considerations show that the gain of the VOR must be greater than one to fully stabilize a visual image. Head movements about axes displaced with respect to the eye cause translation as well as rotation of the eyes in space, and give rise to otolith as well as canal afferent signals. However, the otolith and canal signals are insufficient to determine the correct VOR gain; information about target location is also required. The central goal of this proposal is to identify the site(s) where the computation of VOR gain occurs, and then to analyze the neural pathways and physiological mechanisms involved in producing compensatory eye movements. This work relates to issues of how we determine spatial location, and to neural mechanisms involved in binocular control of eye movements.

DESCRIPTION: (Adapted from the applicant's abstract.) The vestibulo-ocular reflex (VOR) is essential for normal visual acuity because it acts to stabilize visual images on the retina against movements of the head and body. Head and body movements are sensed by the semicircular canals (angular accelerometers) and the otolith organs (linear accelerometers). The shortest neural pathway is a 3-neuron arc consisting of primary receptor neurons, interneurons in the vestibular nuclei, and motoneurons that drive the muscles of the eye. The reflex is extremely fast, exhibiting a latency as short as 8 ms in the monkey. In general, if one is viewing a target in the distance, the compensatory eye movements should be equal in magnitude but opposite in direction to the perturbing head movement (Gain - -1). Frequently, however, one views an object in extrapersonal space, e.g., something held in the hands. For near targets such as these, there is no single correct gain for the VOR, since the axis of head rotation cannot be coincident with the rotational axes of the eyes. Normally the center of rotation is behind the eyes (e.g., above the spinal cord for left or right head turns), and geometric considerations show that the gain of the VOR must be greater than one to fully stabilize a visual image. Head movements about axes displaced with respect to the eye cause translation as well as rotation of the eyes in space, and give rise to otolith as well as canal afferent signals. However, the otolith and canal signals are insufficient to determine the correct VOR gain; information about target location is also required. The central goal of this proposal is to identify the site(s) where the computation of VOR gain occurs, and then to analyze the neural pathways and physiological mechanisms involved in producing compensatory eye movements. This work relates to issues of how we determine spatial location, and to neural mechanisms involved in binocular control of eye movements.

DESCRIPTION (Adapted from applicant's abstract): The vestibular ocular reflex (VOR) is essential for normal vision because it reduces image motion on the retina during head movements. The visual capability of patients with VOR disorders is severely impaired by blurred and double vision. Although clinical tests for semicircular canal dysfunction are available, there are no standard clinical tests for otolith dysfunction and linear VOR deficiencies. Ocular compensation of translational head movements (the linear VOR) is more complex than compensation for rotatory head movements, and its neurophysiology is much less well understood. For the linear VOR, the direction and amplitude of compensatory eye movement depends on gaze direction, viewing distance, and the linear motion of the head. Unlike the angular VOR, there is no fixed relationship between the vestibular inflow and the motor output. The complex relationship of the linear VOR to gaze suggests that it is a behavior somewhere between reflexive and voluntary. The goal of this proposal is to elucidate the neurophysiological basis for the interaction of gaze information with otolith afferent signals that underlies the generation of motor commands to extraocular muscles for the linear VOR. To accomplish this goal, we will use single unit recording to quantitatively analyze central signals encoding linear motion and oculomotor variables in non-human primates. Two hypotheses will be rigorously evaluated. Hypothesis 1: Vestibular neurons that encode eye and head velocity in the same direction (Eye-Head Neurons) are essential components in linear VOR pathways. Eye-Head neurons will exhibit monocular eye movement related activity and encode gaze-modulated linear head movement signals. Hypothesis 2) Purkinje cells, located in the cerebellar flocculus/ventral paraflocculus will encode gaze-modulated linear head movement signals. In both structures, neurons will be organized according to eye movement direction and ocular selectivity. We will identify cells behaviorally, and determine their connectivity with the peripheral labyrinth and/or the cerebellum using electrical microstimulation. Our goal is to determine how networks of these cells transform vestibular afferent signals from the otoliths and central eye movement signals related to gaze into oculomotor commands.

DESCRIPTION (provided by applicant): In mice the lack of an easily administered and selective test for vestibular function is a significant impediment for studies of the vestibular system, especially for those studies that seek to assess quantitatively functional losses associated with aging or the efficacy of drug treatments or genetic manipulations. The overall goal of this proposal is to develop and validate easily used functional vestibular tests for mice to use in such studies. There are 2 specific aims. The first aim is to optimize measurements of the vestibulo-collic reflex (VCR) in animals subjected to en bloc dynamic rotation about earth vertical and earth horizontal axes. Preliminary data suggests the feasibility of this approach, but the extent of the vestibular system's contribution to the response must still be determined. Since mice orient their heads with respect to gravity, we will also develop a, simplified method of measuring head orientation based on an analysis of video recordings of head position during static tilts. These tests are based on the hypothesis that the neck reflexes are dependent on vestibular sensory inflow and act to stabilize head position and orientation during whole body motion in space. Sensory cell losses or peripheral innervation changes should result in measurable changes in vestibular neck reflexes. Aim 2 will assess changes in the static and dynamic VCR in a cohort of aging mice by comparing changes in VCR with changes in peripheral morphology or synaptic organization. C57BL/6 mice aged 12,18, 24 and 30 months will be tested to determine if the efficacy of vestibular neck reflexes declines with age. The vestibular test data will be compared to non-specific methods of assessing vestibular and motor function such as "balance beam and rota-rod tests. The temporal bones of the aged mice will be subjected to morphological analyses to determine if there are age related changes in the numbers of sensory hair cells or hair cells with calyx endings (Type I hair cells).

DESCRIPTION (provided by applicant): This proposal will establish a new research direction and develop novel techniques for my laboratory while promoting a new collaboration between my lab and that of my co- investigator, Dr. Yehoash Raphael. We plan to develop a guinea pig model of vestibular function for two sets of planned studies in the future: first, a study of hair cell regeneration and the physiology that underlies recovery of function in the vestibular system, and second, a study of central vestibular signals related to detection of linear acceleration, which occurs when head orientation with respect to gravity is changed or when the head is translated inertially in space. There are 5 specific aims, all performed in alert guinea pigs: (1) demonstrate and characterize head and eye orientation responses to gravity;(2) characterize the frequency responses of the vestibulo-collic (VCR) and vestibulo-ocular (VOR) reflexes about multiple axes;(3) characterize ocular responses to low and high frequency linear accceleration in the horizontal plane;(4) using methodology developed in Aims 1-3, quantitatively assess the time course and extent of recovery of dynamic and static vestibular reflexes after bilateeral chemical lesions of the vestibular periphery;and (5) determine the time course and extent of recovery, if any, of vestibular reflexes after bilateral surgical labyrinthectomies. Eye and head position in space will be measured using 3D dual search coils and vestibular stimulation will be provided using a servo- controlled turntable that generates angular motion about earth vertical or horizontal axes or a linear sled that accelerates in the horizontal plane. At the completion of lesion experiments, temporal bones will be dissected and analyzed to assess the extent of loss and spontaneous regeneration of sensory hair cells. Recovery of vestibular reflexes will be correlated with the extent of hair cell loss or possible regeneration over time. This project has significant potential to enhance health related research since no therapeutic procedure is currently available to replace vestibular loss despite its serious consequences for affected individuals. Although patients may recover substantially, the extent of recovery depends on age and overall health, and the physiological mechanisms that underlie it are not fully understood. The set of proposed studies will characterize the fundamental vestibular mechanisms involved in spatial orientation and gaze stability and will address the question of how these mechanisms are affected by both chemical and surgical modification of the vestibular end organs.Project Narrative: This project has significant potential to enhance health related research since no therapeutic procedure is currently available to replace vestibular loss despite its serious consequences for affected individuals. Even fully compensated patients may lose their balance and fall in darkness or if exposed to uneven surfaces. Falling behavior, at least partially related to vestibular loss, is also a significant cause of mortality in the elderly.

DESCRIPTION (provided by applicant): Voluntary rapid eye and head movements are used to shift gaze in space. Rapid eye movements are shorter in duration than head movements necessitating compensatory ocular counter-rotation during head turns in order to stabilize the retinal image and direction of gaze in space (equal to the sum of eye and head position). It is widely accepted that the vestibulo-ocular reflex (VOR) produces the compensatory eye movement. However, recently published data demonstrate that when gaze stability is a behavioral goal, guinea pigs use extra-vestibular signals (e.g., efference copy of head movement) in place of the VOR to compensate for active head movement. This response is anticipatory because it occurs with zero latency relative to the head movement. We hypothesize that the extra-vestibular signal is encoded in the vestibular nucleus and/or cerebellum from either an efference copy of the intended head movement or proprioceptive feedback related to the head movement. Since the anticipatory response must replace the VOR, the reafference that results from the active head movement must also be cancelled. We hypothesize that an internal model of the vestibular sensorium is used to transform the extra-vestibular signal into a signal that cancels the reafference. Specifically, we hypothesize a neural circuit that includes a subset of vestibular-only (VO) and eye movement sensitive (ES) neurons in the vestibular nucleus and Purkinje cells in the cerebellar flocculus perform this cancellation and produce the anticipatory eye movement. The specific aims of this proposal are designed (1) to directly test this hypothesis by recording from secondary vestibular neurons of head-unrestrained guinea pigs during passive and active head turns and (2) to refine and demonstrate the effectiveness of two novel devices: a miniature micro-drive and a 6-dof motion sensor to record single unit data and head movements in multiple dimensions from a head unrestrained animal.

? DESCRIPTION (provided by applicant): One of the hallmarks of human evolution is the extraordinary degree to which we can manipulate the physical world with our hands or with tools that extend or amplify portions of our forelimb and digits. This manual dexterity coevolved with an expansion of posterior parietal cortex (PPC), which contains areas involved in programming voluntary movements, coding reach targets in multiple reference frames, and decision making. To allow our body to interact with our physical surroundings, these fields must also construct an internal model of the physical self: our body's configuration, the boundary between our body and external physical objects, and the temporary expansion of that self as we wield a tool that extends our reach and manual capabilities. Such comprehension of where and what the self is and even the ability to manipulate objects and use them as tools did not evolve de novo in humans, but rather emerged from simple networks likely to be present in early mammals. The overarching goal of this proposal is to use a multileveled comparative approach to determine how simple networks associated with reaching and grasping were modified to produce the sophisticated abilities associated with the human condition. In four important animal models (rats, tree shrews, prosimian galagos, and macaque monkeys) we will use electrophysiological recording techniques, intracortical microstimulation (ICMS), and neuroanatomical tracing techniques to define homologous areas in PPC. We, and others, have proposed that PPC generates movements guided by the integration of multisensory inputs occurring in PPC. During movements, neurons in motor areas and movement-specific domains of PPC coordinate their activity to generate unique sequences of body, forelimb and hand postures necessary for context-dependent target acquisition and other movements. This hypothesis will be tested in two ways: (1) We will reversibly deactivate motor (M1) cortex in macaque monkeys and rats and examine the effects on movement domains elicited by ICMS in PPC, and (2) We will reversibly deactivate M1 and cortical areas in PPC in macaque monkeys during a natural, bimanual target acquisition task to reveal how these cortical areas work together to generate accurate and contextually appropriate reaching and grasping. By combining connectional, functional, and behavioral data from multiple species, these studies will provide a rich understanding of the role of these complex brain networks in the planning and execution of movement.

Abstract Vestibular dysfunction is a significant public health problem. Agrawal et al. (2009) reported that 35% of adults older than 40 had evidence of postural instability. Balance dysfunction is linked to an increased likelihood of falling and in the U.S. falls are responsible for more than 50% of accidental deaths. Although the causes of vestibular dysfunction are multiple, recent studies suggest a linkage between noise-induced hearing loss and vestibular dysfunction (Akin et al. 2012; Golz et al. 2001; Guest et al. 2011; Zuniga et al 2012). The suggestion that noise exposure is also a risk factor for vestibular dysfunction is controversial as there is only limited experimental support for causal relationships between noise exposure and peripheral vestibular pathology and behavioral symptoms (e.g., poor balance). In our recently published study (Stewart et al. 2018), we exposed rats to 6 hours of 120dB SPL low frequency noise (3-octave band centered at 1500Hz) and found that neural activity in the vestibular nerve was reduced, as assessed by the vestibular short latency evoked potential (VsEP). Noise exposed animals also exhibited reduced numbers of immunostained afferents with calyx endings, especially calyx-only afferents that terminate on hair cells located in the striolar region of the sacculus (Stewart et al. 2018). More recent experiments show that noise, depending on its intensity, can cause either temporary or permanent threshold shifts of VsEP responses to jerk stimuli. Permanent noise induced VsEP threshold shifts could reflect loss of calyces and/or concomitant loss of ribbon synapses within calyces. This loss might be permanent or there could be recovery associated with reconnection of calyces or recovery of synapses. We hypothesize that noise disrupts peripheral vestibular synapses and/or synaptic transmission, transiently or permanently and causes functional vestibular loss. Determining the basis for synaptic/signal transmission failure in the vestibular periphery and the parameters that characterize damaging noise is a critical first step toward development of future preventative measures. Specific Aim 1 will determine the parameters of noise that causes temporary versus permanent changes to the VsEP and to peripheral vestibular nerve terminals and their synapses in the saccular and utricular maculae. Specific Aim 2 will extend the analysis of Aim 1 to examine the semicircular canal cristae and compare noise-induced changes in the cristae with those observed in the otolith organs. Specific Aim 3 will correlate changes in VsEP responses and noise induced synaptic pathology with behavioral assays: a beam crossing task, an otolith dependent behavior (macular ocular reflex, MOR), and a semicircular canal dependent behavior (vestibuloocular reflex, VOR).