Nicotinic acetylcholine α4β2∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4β2∗ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4β2∗ nAChRs in [3H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4β2∗ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4β2∗ nAChRs, labeled with antibodies specific for a β2 subunit extracellular epitope indicating that nifrofam labels α4β2∗ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4β2∗ nAChRs in brain regions.

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pK_a and high affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rs in vitro Nicotine, with lower pK_a and α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in male and female mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET ¹⁸F-labeled imaging ligands were chosen: [¹⁸F]2-FA85380 (2-FA) with varenicline-like pK_a and affinity and [¹⁸F]Nifene with nicotine-like pK_a and affinity. [¹⁸F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4β2R-containing GSats. In contrast, [¹⁸F]Nifene kinetics were rapid, consistent with its binding to α4β2Rs but no trapping. Specific [¹⁸F]2-FA and [¹⁸F]Nifene signals were eliminated in β2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on β2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [¹⁸F]2-FA distributions while having little effect on [¹⁸F]Nifene distributions in vivo consistent with only [¹⁸F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4β2R-containing GSats, which results in very slow release long after nicotine is gone after smoking.SIGNIFICANCE STATEMENT Mechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in α4β2R-containing acidic vesicles. Using a fluorescent-labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were α4β2R-containing Golgi satellites (GSats). In vivo PET imaging with F-18-labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.