Development of dopamine D2 receptor-targeted DARTs

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Alterations in dopamine neurotransmission have been implicated in various neuropsychiatric and neurodegenerative disorders, including schizophrenia, depression, attention-deficit hyperactivity disorder, Parkinson’s disease, and substance use disorder. Distortions in timing and anticipation are symptoms, as well as a source of additional complications, in many of these disorders. Neuroimaging studies in humans, as well as a wide range of animal studies, have shown that dopaminergic modulation o f cortico-striatal-thalamic circuits alters timing and that both D2 and D1 receptor signaling are important for accurate and precise temporal control. A major barrier to understanding the intricacies of drug action in vivo relates to the complex and manifold localization of the same receptor throughout the brain. For example, in the striatum, dopamine D2 receptors (D2Rs), are located on indirect pathway medium spiny neurons (iMSNs), cholinergic interneurons, the terminals of dopaminergic projections from the midbrain, as well as on glutamatergic corticostriatal terminals. Given this complexity, it can be impossible to infer the precise action of dopamine at specific D2Rs, or the consequences of blocking this action by antagonists, as the same type of receptor can have complementary or opposing effects on circuit function when present in different neurons, or even in different locations within the same neuron. Local intracranial injections of drugs have been used as a strategy to differentiate the local actio ns of drugs from their systemic effects, but in a complex region such as the striatum, this is not adequate to differentiate actions of D2Rs expressed on different neuronal subtypes. While Cre-dependent knockout of D2R can begin to address these issues, limitations of this approach include developmental effects, compensation, and the fact that D2Rs outside of the striatum are also deleted, making the effects complex to interpret. Drugs Acutely Restricted by Tethering (DARTs) is a new approach that allows targeting of endogenous receptors with cell-type specificity using direct pharmacological treatment. This strategy has been used successfully to target ionotropic glutamate receptors in specific neuronal populations in vivo and has also been debuted in vitro for a muscarinic acetylcholine GPCR but has not yet been developed for studying GPCRs in vivo. Importantly, the DART approach has the advantage that the activity of unmodified, natively expressed receptors can be controlled. Leveraging collaborative expertise in chemistry, protein engineering, genetic engineering of mice, and behavioral analysis, we propose to use this method to interrogate D2R function in vivo, with an initial focus on identifying the neuronal cell types in the striatum in which D2Rs regulate timing. We have had initial success in developing D2R-targeted DARTs, which we have validated both in vitro and in vivo and thus propose the following aims: Aim 1: Optimize DARTs for cell-type-specific dopamine D2 receptor antagonism in awake behaving animals. Aim 2: Use DARTs to identify the neuronal population in the dorsal striatum in which dopamine D2 receptors modulate timing.