ANTISENSE KNOCKOUT OF CNS D2 DOPAMINE RECEPTORS

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Despite the fact that dopaminergic receptor agonists and antagonists are standard therapy for a number of psychiatric and neurological disorders, study of the neurobiology of dopamine receptors is severely hampered by an inability to selectively manipulate individual dopamine receptor subtypes. Pharmacological tools permit only two receptor "families" (D1 and D2) to be distinguished whereas recent cloning studies demonstrate the existence of at least 5 dopamine receptor subtypes. The present experiments capitalize on these recent molecular advances by utilizing antisense oligodeoxynucleotides to selectively block the production of dopamine receptors in vivo. Antisense knockout allows the highly specific arrest of mRNA translation into functional receptor protein providing, for the first time, a selective means for acquiring functional information regarding individual dopamine receptor subtypes. Preliminary data are included to show that CNS infusion of D2 antisense oligo produces specific D2 dopamine receptor knockout in rat brain along with decreases in dopamine-mediated behaviors and autoreceptor function. We will use receptor autoradiography and homogenate binding to effect a parametric analysis of D2 dopamine receptor knockout in terms of time course, dose-relatedness, and receptor specificity. Local, intracerebral application of the antisense oligo will allow for selective knockout of post-versus pre-synaptic D2 receptors. Functional aspects of D2 dopamine receptor knockout will be determined by analysis of spontaneous and drug-induced motor activity. In vivo microdialysis will be used to assess the impact of specific D2 knockout on striatal acetylcholine release and on autoregulation of the release and synthesis of striatal dopamine. In vivo electrophysiological experiments will be used to determine the dopamine receptor subtype responsible for autoreceptor-mediated changes in dopamine terminal excitability, and to monitor any effects of antisense treatment on dopamine neuron spontaneous activity. Finally, we propose to apply the in vivo antisense technique to the study of other dopamine receptor subtypes, beginning with the D3 receptor for which pilot data is presented. Unique information on the role(s) of specific dopamine receptor subtypes in the central actions of dopamine will be obtained from these studies. The data are relevant to understanding the role of dopamine in normal and abnormal behavior and to the development of more specific ways to influence these processes.