Neal Barmack, Ph.D. - US grants

Partial recovery of function following a unilateral labyrinthectomy (UL), termed "vestibular compensation," has served as a model for investigations of subcellular investigations of central nervous system plasticity. Previous studies of compensation have "targeted" particular genes for study, using immunohistochemical and hybridization histochemical probes. Rather than target a particular genes, we propose to use the technique of "differential display" (DD-PCR) to screen gene products isolated from vestibular-and auditory-related regions the rabbit brain. Unlike the "targeted gene" approach, DD-PCR screens all differentially transcribed gene products. First, we will screen Scarpa's ganglion, medial vestibular nucleus, nodulus and inferior colliculus following UL. Since the primary vestibular-auditory afferent projects to the nodules and the DCN are unilateral, the structures on the side of the UL will receive a decreased primary afferent input and those on the intact side will receive a normal input. Second, we will use oligonucleotide probes identified by DD-PCR to reveal the tissue distribution and the time course of changes in tissue distribution of different mRNAs in animals that have received a UL. Third, promising molecules will be studied under more physiological conditions. Maintained static tilt will be used to provide asymmetric vestibular stimulation. Unilateral removal of the ossicular chain will be used to create asymmetric acoustic stimulation. Horizontal optokinetic stimulation will be used to provide asymmetric visual climbing fiber inputs to the nodulus. The optokinetic inputs will allow us to screen molecules that are differential expressed in the same structure, the nodulus, under stimulus conditions mediate db two different afferent pathways. Fourth, in analogous screening experiment at the protein level we will use two-dimensional electrophoresis to screen for proteins that are differential expressed following UL. Fifth, some molecules may participate in compensation or plasticity merely by changing their distribution within a cell rather than by changing expression. We will examine such a molecule, PKC-delta, in "activated" Purkinje cells using combined physiological, immunohistochemical and ultrastructural methods. Gene products uncovered by these experiments might play a role in both vestibular and auditory adaptation and provide important clues for the pharmacological treatment of central neural disorders such as motion sickness and tinnitus.

DESCRIPTION (provided by applicant): We propose three areas of investigation to understand how the circuitry of the vestibulocerebellum uses vestibular information to modify the simple spike (SS) output of Purkinje cells and how this output contributes to behavioral adaptation to altered vestibular orientation: 1) Circuitry transmitting vestibular information to inferior olive. The parasolitary nucleus (Psol) conveys vestibular signals to the beta-nucleus and dorsomedial cell column (dmcc), two subnuclei of the inferior olive. Unilateral lesions of Psol reduce, but do not eliminate vestibularly-modulated climbing fiber responses (CFRs) in the contralateral uvula-nodulus, implying an alternative presynaptic vestibular pathway to the inferior olive. The Y-group may be the origin of this alternative pathway. We will combine retrograde and orthograde tracers with recordings from single Y-group neurons to determine the types of vestibular and optokinetic signals carried by them. 2) Conjunctive CFR-SS vestibular plasticity in the nodulus, understanding interactions between climbing and parallel fibers on Purkinje cells is critical to understanding the cerebellum. Interactions occur with short- or long-term consequences. We will electrically stimulate parallel and climbing fibers in vivo so that their interactions can be interpreted in terms of known vestibular circuitry. A climbing fiber volley from a known semicircular canal zone in the beta-nucleus, will be interacted with a parallel fiber volley from a single vertical semicircular canal. These interactions should reveal how climbing fiber topography is reflected in Purkinje cell output. 3) Behavioral evaluation of the role of the uvula-nodulus in the dynamic control of vestibule-ocular reflex orientation in space. Rotation of rabbits about a longitudinal axis modulates the velocity and orientation of a previously induced optokinetic afternystagmus (OKAN II). The significance of decreases in nystagmus velocity and the possible contribution of the nodulus to modification of eye velocity are poorly understood. We suggest that head tilt induces a reduction in nystagmus velocity to reduce the gain of all eye reflexes that conflict perceived vertical orientation or a previously "remembered" orientation. We will measure changes in slow phase velocity when the orientation of the rabbit's head causes nystagmus to be executed at different angles within the orbit before and after bilateral destruction of the uvula-nodulus.

DESCRIPTION (provided by applicant): In vivo analysis of cerebellar circuitry has focused almost exclusively on Purkinje cells, identified by their iconic patterns of complex and simple spikes (CSs and SSs). However, views of cerebellar function based exclusively on the physiology of Purkinje cells ignore the role of interneurons and distort the attributes of cerebellar afferent systems. It is universally assumed that SSs are modulated by the activity of the mossy fiber-granule cell-parallel fiber projection to Purkinje cell dendrites. In fact, during natural vestibular stimulation, vestibular primary afferent mossy fiber afferents discharge out of phase with the SSs recorded from nodular Purkinje cells. Consequently, it is unlikely that the cerebellar output signal merely reflects a gain-controlled version of the mossy fiber input signal. We proposed that SS modulation reflects the action of climbing fibers on cerebellar interneurons. We will test specific versions of this hypothesis by recording from identified interneurons. We have three objectives. First, we will record extracellularly from interneurons in the uvula-nodulus of anesthetized mice during natural vestibular stimulation. Interneurons will be labeled juxtacellularly with neurobiotin. The depth and phase of modulation of interneuronal discharge relative to that of Purkinje cell CSs and SSs will indicate which interneurons could modulate SSs. Second, we will study how the modulated activity of interneurons and Purkinje cells is altered by a unilateral labyrinthectomy (UL). Following a UL, the ipsilateral uvula-nodulus is accessible to vestibular information mediated only by climbing fibers whose modulation depends on the contralateral, intact labyrinth. Third, we will also make microlesions in the p-nucleus and dorsomedial cell column (dmcc) in the contralateral inferior olive. This will leave one side of the cerebellum accessible to vestibular information mediated only by vestibular mossy fibers. We will compare the effects of reduced vestibular signaling on interneurons and Purkinje cells. Fourth, we will microinject miRNAs in viral vectors with a cell specific promoter to selectively reduce expression of GABA-A alpha 1 receptors in nodular Purkinje cells. Fifth, we will also use miRNAs to selectively reduce synthesis of GABA in Golgi cells. We will analyze the effects of "knocking down" GABAergic signaling in these two cell types. We will characterize the stimulus-modulated functions of identified interneurons for the first time. We will interfere with cerebellar circuitry at a cellular level and test the role of interneurons in the modulation of SSs. The proposed research will speed application of molecular techniques to the treatment of patients with cerebellar disorders.