The purpose of the proposed investigation is to determine the mechanism by which changes in neuronal activity of dopamine (DA)-containing neurons of the mammalian nervous system influence the regulation of tyrosine hydroxylase (TH). The short-term and long-term regulation of TH will be studied, and the functional significance of multiple forms of TH will be investigated. The role of phosphorylation of TH in the activation of this enzyme that occurs in response to increases in neuronal activity will be investigated. The effects of several classes of protein kinases on TH of retina and corpus striatum will be investigated and compared to the activations of TH produced in vivo as a consequence of increased neuronal activity. The ability of phosphoprotein phosphatases to inactivate TH in vitro following activation of TH in vivo will be examined, and the effects of in vivo activation of TH on subsequent phosphorylation of the enzyme in vitro with [32P]-ATP will be determined. We will determine if the incorporation of 32P-phosphate into retinal TH occurs in situ as a consequence of exposure to light, a physiological stimulus that increases the rate of DA synthesis and release, and activates TH in retinal DA-containing neurons. Experimental evidence indicates that the regulation of retinal TH in response to prolonged, tonic changes in neuronal activity occurs by different mechanisms than that in response to short-term changes. We will, therefore, compare the long-term regulation of TH in retina with that in axon terminal and somatodendritic areas of the mesolimbic and mesocortical DA-containing neurons. To further investigate the mechanisms underlying the short- and long-term regulation of DA synthesis, we will separate and characterize the multiple molecular forms of TH, and investigate the mechanisms involved in the interconversion of the various forms. The results of these studies will significantly increase our knowledge of the mechanisms involved in the regulation of DA synthesis and release in response to both short-term and long-term changes in neuronal activity. Because DA appears to play a role in many physiological processes and neurological/neuropsychiatric disorders, these studies have broad significance to the neurosciences.
