Abstract
Immunolabeling and autoradiography have traditionally been applied as the methods-of-choice to visualize and collect molecular information about physiological and pathological processes. Here, we introduce PharmacoSTORM super-resolution imaging that combines the complementary advantages of these approaches and enables cell-type- and compartment-specific nanoscale molecular measurements. We exploited rational chemical design for fluorophore-tagged high-affinity receptor ligands and an enzyme inhibitor; and demonstrated broad PharmacoSTORM applicability for three protein classes and for cariprazine, a clinically approved antipsychotic and antidepressant drug. Because the neurobiological substrate of cariprazine has remained elusive, we took advantage of PharmacoSTORM to provide in vivo evidence that cariprazine predominantly binds to D3 dopamine receptors on Islands of Calleja granule cell axons but avoids dopaminergic terminals. These findings show that PharmacoSTORM helps to quantify drug-target interaction sites at the nanoscale level in a cell-type- and subcellular context-dependent manner and within complex tissue preparations. Moreover, the results highlight the underappreciated neuropsychiatric significance of the Islands of Calleja in the ventral forebrain.
Highlights
Immunolabeling and autoradiography have traditionally been applied as the methods-ofchoice to visualize and collect molecular information about physiological and pathological processes
We applied a generalizable workflow for the production and validation of a PharmacoSTORM probe for one of the most abundant G protein-coupled receptors in the brain, the CB1 cannabinoid receptor (CB1R) (Fig. 1a)
In this study, we introduce a general approach for the application of pharmacological probes to localize proteins at the nanoscale level and developed a pipeline to visualize drug–target interactions in a cell- and subcellular compartment-specific manner in complex tissue preparations
Summary
Immunolabeling and autoradiography have traditionally been applied as the methods-ofchoice to visualize and collect molecular information about physiological and pathological processes. Because the neurobiological substrate of cariprazine has remained elusive, we took advantage of PharmacoSTORM to provide in vivo evidence that cariprazine predominantly binds to D3 dopamine receptors on Islands of Calleja granule cell axons but avoids dopaminergic terminals. These findings show that PharmacoSTORM helps to quantify drug-target interaction sites at the nanoscale level in a cell-typeand subcellular context-dependent manner and within complex tissue preparations. Antibodies can label immature or inactive proteins their usefulness to quantify functionally relevant pools of signaling molecules is limited These methodical obstacles together introduce substantial technical variability and may even contribute to erroneous diagnostic interpretations in the clinical use of immunostaining[13]. Small molecules readily penetrate tissue preparations and bind their targets with known stoichiometry
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