Georgia Ann Bishop - US grants

The output of the inferior olive, described as climbing fibers in the cerebellar cortex, has been implicated as playing a critical role in "cerebellar learning". Changes in olivary output result in significant alterations in the firing pattern of Purkinje cells in the cerebellar cortex. The nature of olivary output is modified by multiple afferent systems and by local inhibitory circuits which have been postulated to play a role in regulating the synchronous firing of olivary neurons. Further, it has been shown that serotonin (5HT) may be the neurochemical mediator of local inhibitory circuits. However, in addition to 5HT at east two other putative inhibitory neurotransmitters have been identified-enkephalin (ENK) and gamma aminobutyric acid (GABA). The specific aims of this proposal are: 1) to compare the distribution of 5HT, ENK and GABA in the inferior oolivary complex of the rat using the indirect peroxidase antiperoxidase (PAP) technique of Sternberger (13); 2) to identify the cell bodies of origin for these putative neurotransmitters to the inferior olivary complex using a double labeling technique which combines retrograde transport of horseradish peroxidase with the PAP technique in order to identify the putative neurotransmitters used by reticulo-olivary neurons; and 3) to determine the physiological and morphological characteristics of serotoninergic neurons in the medullary reticular formation, especially those which participate in the local circuitry of the inferior olivary complex, using a double labelling technique which combines intracellular recording and staining of individual neurons with fluorescent immunohistochemical techniques (30-34). These neurons will be analyzed at the light and electron microscopic level in order to determine the nature of their interaction in the olivary coplex. In summary, the manner in which information is processed within a nucleus has a direct relationship on its output. In the olivo-cerebellar circuit, suble changes in climbing fiber input may result in significant alterations in Purkinje cell responsiveness and ultimately in cerebellar output. Thus, data obtained in this project should extend our knowledge of the role local circuits in the inferior olivary complex play in cerebellar mediated movements.

The intent of this proposal is to determine the distribution, origin and physiological effect(s) of two putative neuromodulators, the indoleamine serotonin and the peptide enkephalin, on Purkinje cells in the cat's cerebellum. The Specific Aims of this proposal are: 1) To determine the lobular and laminar distribution of serotonin and enkephalin in the cat's cerebellum using the peroxidase anti-peroxidation technique. 2) To localize the cells of origin of serotoninergic and enkephalinergic varicosities present within the cerebellum. This will be accomplished by using a double label technique which combines retrograde transport of horseradish peroxidase (HRP) with the PAP technique. 3) To analyze the physiological effects of serotonin and enkephalin on Purkinje cells. Various putative neuroactive substances (e.g. serotonin, enkephalin, glutamate, GABA) will be iontophoresed from multibarrel extracellular recording electrodes and their on Purkinje cell activity recorded. In some experiments an intracellular electrode will be combined with the drug electrode. The location of each unit will be marked either with Fast Green (extracellular recording) or HRP (intracellular recording). The tissue will be processed with the PAP technique to verify the presence of serotoninergic and enkephalinergic varicosities in areas from which physiological data are obtained. 4) To establish the relationship of serotoninergic and enkephalinergic varicosities to other axonal elements within the cat's cerebellum. The spatial relationship of immunoreactive varicosities to Purkinje cells intracellularly injected with HRP will be analyzed. In addition, the cytological characteristics or terminals containing serotonin and enkephalin as well as their synaptic relationships will be determined. The role of these chemically defined systems has not been incorporated into existing theories of cerebellar function as little is known about their effect(s) on specific populations of cortical neurons. It has been proposed that neuromodulators play a role in regulating neuronal activity and that they function to bias or fine tune neural circuits which, ultimately, influence on-going- movements. Thus, data from the proposed studies, when incorporated into our existing concepts of cerebellar physiology, will further extend our knowledge of how individual circuits in the cerebellar cortex contribute to the control and co-ordination of movement.