The plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) are the main sites of action for therapeutic and abused stimulant drugs. As a means to identify novel medications for stimulant addiction and other psychiatric disorders, we developed in vitro assays in rat brain tissue that can be used to determine structure–activity relationships for test compounds at these monoamine transporters. Uptake inhibition assays measure the ability of drugs to block the transporter-mediated uptake of [3H]neurotransmitters into synaptosomes, whereas release assays measure the ability of drugs to serve as transporter substrates that evoke efflux (i.e., release) of [3H]neurotransmitters from synaptosomes by reverse transport. These assays can be used to rapidly determine the potency of test compounds at DAT, NET, and SERT under similar conditions, establishing the selectivity of drugs across all three transporters. The combined results from uptake and release assays can discriminate whether a compound is a transporter inhibitor or substrate (i.e., releaser). Our assay procedures have been used to characterize the molecular mechanism of action for older amphetamine-type medications and newer transporter ligands with therapeutic potential. The data from these assays can also predict the addictive and neurotoxic properties of abused stimulants. Information provided by these assays continues to provide insight into monoamine transporter structure and function.
Interrogating the Activity of Ligands at Monoamine Transporters in Rat Brain Synaptosomes