Jay M. Goldberg - US grants

Microelectrode studies are done in the squirrel monkey and the chinchilla with the purpose of understanding the functioning of the labyrinth in terms of the overall operation of the vestibular system. Four interrelated projects are proposed. I) A combined morphological and physiological approach is used to study the relation between the discharge properties of vestibular-nerve afferents and peripheral innervation patterns in the chinchilla, as well as the cellular mechanisms of repetitive discharge and synaptic transmission within the sensory epithelium. II) An intracellular, electrophysiological paradigm shows that individual secondary neurons in the monkey receive different proportions of their monosynaptic, vestibular-nerve input from regularly and irregularly discharging afferents. To understand the functional significance of these differential projections, they are being correlated with the central connections and locations of secondary neurons determined by a combination of electrophysiological and intracellular dye-injection techniques. III) The head-velocity signal carried by secondary canal-related neurons, which differ from one unit to another, may be related to the distinctive vestibular-nerve inputs, regular or irregular, that each of them receives. The discharge of the secondary neurons are to be recorded in the alert monkey and criteria developed to deduce the profiles of vesticular-nerve inputs by conventional extracellular techniques. IV) Single-unit studies will be done in the alert monkey to investigate possible efferent-induced responses of vestibular-nerve afferents to head and neck rotations. The discharge of brain-stem efferent neurons will be recorded, first in decerebrate monkeys and then, possibly, in the alert animal.

Our goal is to understand the functional organization of the labyrinth in terms of the overall operation of the vestibular system. Recent morphophysiological studies done in our laboratory have established a relation between the peripheral innervation of a vestibular afferent and its discharge characteristics. Specifically, three physiological classes of afferents were recognized: regular units, irregular high-gain units and irregular low-gain units. The first class corresponds to dimorphic (and bouton) units, the second to dimorphic units, and the third to calyx units. We are now interested in determining the function of the various classes of afferents, which can only be done by ascertaining how the brain makes use of the information provided by them. The approach is to use morphophysiological techniques in the chinchilla to trace the central trajectories of the three afferent classes and, thus, ascertain if they differ in the topography of their projections to the vestibular nuclei, other brainstem sites and the cerebellum or in the morphology of their axonal branches and terminals. Axons are impaled near their entrance into the vestibular nuclei, they are physiologically characterized, and then injected with horseradish peroxidase (HRP). After suitable histochemical processing, the central and peripheral trajectories are reconstructed. A single, physiologically characterized afferent is injected in each nerve. The experiment is repeated until a sample of approximately 20 axons from each of the three classes is obtained. The data should provide a detailed picture of the projection patterns of each class, as well as the central branching and innervation patterns of individual axons. A similar analysis will be done for all five endorgans. To provide a context for our morphophysiological data, anatomical tracer studies will be done on the commissural, vestibulo-ocular, vestibulospinal and vestibulocerebellar pathways in the chinchilla. The results should provide insights into the functional organization of the labyrinthine receptors and of their central targets and into the evolution of the vertebrate labyrinth. In this last respect, the calyx unit and the Type I hair cells in innervates are of particular interest. These structures are recent phylogenetic acquisitions, first making their appearance in reptiles and then becoming distinctive features of the labyrinth of birds and mammals. An understanding of the function of the calyx unit should shed light on its evolutionary significance.

Our goal is to understand the functional organization of the labyrinth in terms of the overall operation of the vestibular system. Recent morphophysiological studies done in our laboratory have established a relation between the peripheral innervation of a vestibular afferent and its discharge characteristics. Specifically, three physiological classes of afferents were recognized: regular units, irregular high-gain units and irregular low-gain units. The first class corresponds to dimorphic (and bouton) units, the second to dimorphic units, and the third to calyx units. We are now interested in determining the function of the various classes of afferents, which can only be done by ascertaining how the brain makes use of the information provided by them. The approach is to use morphophysiological techniques in the chinchilla to trace the central trajectories of the three afferent classes and, thus, ascertain if they differ in the topography of their projections to the vestibular nuclei, other brainstem sites and the cerebellum or in the morphology of their axonal branches and terminals. Axons are impaled near their entrance into the vestibular nuclei, they are physiologically characterized, and then injected with horseradish peroxidase (HRP). After suitable histochemical processing, the central and peripheral trajectories are reconstructed. A single, physiologically characterized afferent is injected in each nerve. The experiment is repeated until a sample of approximately 20 axons from each of the three classes is obtained. The data should provide a detailed picture of the projection patterns of each class, as well as the central branching and innervation patterns of individual axons. A similar analysis will be done for all five endorgans. To provide a context for our morphophysiological data, anatomical tracer studies will be done on the commissural, vestibulo-ocular, vestibulospinal and vestibulocerebellar pathways in the chinchilla. The results should provide insights into the functional organization of the labyrinthine receptors and of their central targets and into the evolution of the vertebrate labyrinth. In this last respect, the calyx unit and the Type I hair cells in innervates are of particular interest. These structures are recent phylogenetic acquisitions, first making their appearance in reptiles and then becoming distinctive features of the labyrinth of birds and mammals. An understanding of the function of the calyx unit should shed light on its evolutionary significance.

The present proposal addresses two questions; 1) What are the cellular mechanisms determining the discharge properties of vestibular-nerve afferents? and 2) What are the distinctive contributions of the various kinds of afferents to the central processing of vestibular information? The two questions provide complementary perspectives on the vestibular nerve, since to understand a particular class of afferents requires knowledge of both the cellular mechanisms responsible for its discharge patterns and the use made of these patterns by the brain. The present proposal consists of four interrelated projects. Project 1 uses light- and electron-microscopy to investigate the regional organization of the vestibular organs in the chinchilla and the squirrel monkey. The goal is to relate the physiology of afferents with their afferent and efferent synaptic inputs. This is accomplished, in part, by ultrastructural reconstruction of labeled afferents, including some that have been physiologically characterized. Project 2 is a biophysical analysis of transduction mechanisms in the posterior crista of the turtle. This animal was chosen because we have had success in developing the required in vitro preparations. Also, the structural organization of the end organs suggests that the distinctive role of type I hair cells may be easier to discern in turtles than in mammals. The choice requires that we do morphological and physiological studies that parallel those already done in mammals. The biophysical studies will be done in an isolated epithelium, where normal anatomical relations can be maintained, and in solitary hair cells, where precise control of the extracellular and intracellular conditions can be maintained. In project 3, advantage is taken of recent advances in intracellular labeling technology to trace the central trajectories of individual, physiologically identified vestibular-nerve fibers from their origin in each of the five and organs in the chinchilla to their terminations in the vestibular nuclei, other brain-stem sites, and the cerebellum. In this way, we can ascertain if the various afferent classes differ in the spatial distribution of their endings or in the morphology of their axonal branches and terminals. The results should provide the anatomical basis for the segregation and convergence of different classes of afferent inputs in central pathways. Project 4 makes use of a recently devised method for rapidly and reversibly eliminating the discharge of irregularly discharging afferents in the alert squirrel monkey. The resulting functional ablation will be used to assess the contributions of these afferents to the overall operation of the angular and linear vestibulo-ocular reflexes, as well as to the discharge properties of secondary neurons, including those that are likely to contribute to the reflexes. The research program may provide insights into the etiology of vestibular disorders.

The long-term goal of this work is to understand how vestibular organs work. The proper function of these organs is crucial to a healthy existence; damage can lead to debilitating vertigo, dizziness and inability to maintain study gaze. Mamammal, birds and reptiles have similar vestibular organs, with two classes of sensory receptor cell, the type I and type II hair cells. These cells transduce head movements into electrochemical signals that are transmitted across synapses to the terminals of afferent nerve fibers, which convey the signals to the brain in the form of electrical discharges. Efferent nerve fibers from the brain make synapses on h air cells and afferent nerve terminals, through which they influence afferent signals by unknown mechanisms. The specific aims are to characterize: 1) afferent synaptic transmission from the hair cells to the neurons; 2) the cellular mechanisms responsible for discharge regularity and maximum evoked discharge rates of afferent neurons; 3) efferent actions. In vitro preparations of the posterior semicircular canal organ of the turtle will be used. This organ lends itself to comparison of type I and type II hair cells, shows richly diverse efferent actions on afferent nerve fiber discharges, and is robust in vitro. Depending on the specific experiment, stimuli will be mechanical (displacement of the canal fluid), manipulations of membrane voltage or current in hair cells or afferent neurons, or electrical stimulation of efferent nerve fibers. The membrane voltage or current responses of hair cells and afferent neurons to these stimuli will be recorded intra cellularly with sharp micropipettes or patch pipettes. Both conventional (vesicular, orthograde) and unusual forms of transmission between the type I hair cell and afferent neuron will be characterized. Whether afferent discharge regularity is due to presynaptic (hair cell) or postsynaptic (afferent neuron) mechanisms will be tested. whether stages following mechanoelectrical traduction determine saturation of afferent discharge rates will be investigated. Efferent-evoked synaptic potentials and the neurotransmitter receptors responsible will be characterized.

Vestibular organs are provided with a rich efferent innervation, originating in the brain stem. The long-term objective of this study is to understand the function of the efferent vestibular system (EVS) in the processing of vestibular information. There are three specific aims: 1) To determine the effects of electrical activation of different efferent contingents on the several afferent groups in the barbiturate- anesthetized chinchilla. The problem was originally investigated almost 20 years ago. There is a need to update results by taking into account recent findings on the organization on afferent and efferent pathways, as a recent suggestions that EVS stimulation may have more heterogeneous influence on afferent gain than had been suspected. This study provides essential background information concerning the function of the EVS. 2) To study potential long-term or trophic efferent influences by comparing the acute and chronic effects on afferent discharge on eliminating the efferent innervation or the contralateral labyrinth. The presence of neuroactive peptides and metabotrophic receptors, as well as certain features of the response to electrical stimulation suggests that the EVS exerts such an influence, yet the problem has not been previously addressed. 3) To determine the conditions leading to the discharge of efferent neurons and to efferent-mediated changes in afferent transmission. Recordings will be made from vestibular-nerve afferents and from efferent neurons within the brain stem in alert squirrel monkeys free to move their heads in a horizontal plane. For both groups of neurons, responses to active and passive head movements will be compared. The possibility that efferents get inputs from the neck, from other proprioceptors or from several vestibular organs will be explored. Some information on vestibular inputs to the EVS are best obtained in acute preparations; these experiments will be done in decerebrate chinchillas.