In an effort to develop selective radioligands for in vivo imaging of neuronal nicotinic acetylcholine receptors (nAChRs), we synthesized 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380) and labeled it with 125 I and 123 I. Here we present the results of experiments characterizing this radioiodinated ligand in vitro. The affinity of 5-[ 125 I]iodo-A-85380 for ␣4␤2 nAChRs in rat and human brain is defined by K d values of 10 and 12 pM, respectively, similar to that of epibatidine (8 pM). In contrast to epibatidine, however, 5-iodo-A-85380 is more selective in binding to the ␣4␤2 subtype than to other nAChR subtypes. In rat adrenal glands, 5-iodo-A-85380 binds to nAChRs containing ␣3 and ␤4 subunits with 1/1000th the affinity of epibatidine, and exhibits 1/60th and 1/190th the affinity of epibatidine at ␣7 and muscle-type nAChRs, respectively. Moreover, unlike epibatidine and cytisine, 5-[ 125 I]iodo-A-85380 shows no binding in any brain regions in mice homozygous for a mutation in the ␤2 subunit of nAChRs. Binding of 5-[125 I]iodo-A-85380 in rat brain is reversible, and is characterized by high specificity and a slow rate of dissociation of the receptor-ligand complex (t 1/2 for dissociation ϳ2 h). These properties, along with other features observed previously in in vivo experiments (low toxicity, rapid penetration of the blood-brain barrier, and a high ratio of specific to nonspecific binding), suggest that this compound, labeled with 125 I or 123 I, is superior to other radioligands available for in vitro and in vivo studies of ␣4␤2 nAChRs, respectively.Nicotinic acetylcholine receptors (nAChRs) are excitatory ligand-gated cation channels that are widely distributed in mammalian organisms, appearing in the central and peripheral nervous systems, neuromuscular junctions, and adrenal glands. The nAChR channel complex is composed of five protein subunits, which form a pore that is permeable to Na ϩ , K ϩ , and Ca 2ϩ (Lindstrom, 1995;Holladay et al., 1997).To date, ␣, ␤, ␥, ␦, and ⑀ subunits have been isolated and cloned from mammalian and avian tissues, with nine varieties of ␣ and four varieties of ␤ subunits identified. The ␣1, ␤1, ␥, ␦, and ⑀ subunits form the neuromuscular junction receptor, the very first nAChR to be characterized. The other subunits (␣2-␣9 and ␤2-␤4) are found predominantly throughout the nervous system (Lindstrom, 1995;Holladay et al., 1997). This subunit diversity affords a large potential for a variety of nAChR subtypes, exhibiting distinct cationconducting properties and pharmacological heterogeneity. Based on binding properties and pharmacological sensitivity, major nAChR subtypes in mammalian brain can be categorized as ␣-bungarotoxin-sensitive (␣7) and ␣-bungarotoxin-insensitive (e.g., ␣4␤2) (Lindstrom, 1995;Holladay et al., 1997). Accordingly, 125 I-␣-bungarotoxin has been the radioligand of choice for in vitro characterization of the ␣7 subtype of nAChR, whereas tritiated agonists, such as nico-

Assays of human postmortem brain tissue have revealed that smokers have greater densities of high-affinity nicotinic acetylcholine receptors (nAChRs) in several brain regions than do nonsmokers or exsmokers. Quantitative PET imaging of nAChRs in humans has recently been reported using the a4b2* subtypespecific radioligand 2-18 F-FA-85380 (2FA). Methods: We used PET and 2FA to measure total volumes of distribution corrected for the free fraction of 2FA in plasma (V T /f P ) in 10 nonsmokers and 6 heavy smokers (.14 cigarettes/d; abstinent for .36 h). Dynamic PET scans were performed over 8 h, commencing immediately after a bolus injection of 2FA. Anatomic sampling was performed on PET images that were coregistered to MR images acquired from each volunteer. Data were analyzed by Logan plots and by 1-and 2-tissue-compartment models using unbound, unmetabolized arterial 2FA concentration as the input function. Results: All modeling methods yielded similar results. V T /f P was significantly higher in smokers than in nonsmokers in all brain regions tested, except the thalamus. We used measures of V T /f P and estimates of nondisplaceable volume of distribution and found 25%-200% higher values in smokers than in nonsmokers for the volume of distribution for the specific binding compartment in the frontal cortex, midbrain, putamen, pons, cerebellum, and corpus callosum. These findings were consistent with voxel-based analysis using statistical parametric mapping. Conclusion: Our findings suggest that PET with 2FA can be used to study the role of nicotine-induced upregulation of nAChRs in active smokers and during smoking cessation.

N,N-dimethyltryptamine (DMT) is a hallucinogen found endogenously in human brain that is commonly recognized to target the 5-hydroxytryptamine 2A receptor or the trace amine–associated receptor to exert its psychedelic effect. DMT has been recently shown to bind sigma-1 receptors, which are ligand-regulated molecular chaperones whose function includes inhibiting various voltage-sensitive ion channels. Thus, it is possible that the psychedelic action of DMT might be mediated in part through sigma-1 receptors. Here, we present a hypothetical signaling scheme that might be triggered by the binding of DMT to sigma-1 receptors.

Noninvasive imaging of nicotinic acetylcholine receptors (nAChRs) in the human brain in vivo is critical for elucidating the role of these receptors in normal brain function and in the pathogenesis of brain disorders. Here we report the first in vivo visualization of human brain areas containing nAChRs by using PET and 2-[18F]fluoro-3-(2(S)azetidinylmethoxy)pyridine (2-[18F]FA). We acquired scans from six healthy non-smoking volunteers after i.v. bolus administration of 2-[18F]FA (1.6 MBq/kg or 0.043 +/- 0.002 mCi/kg). This dose was sufficient for visualizing nAChRs in the thalamus up to 5 h after injection. There were no adverse effects associated with administration of no-carrier-added 2-[18F]FA (1.3-10 pmol/kg). Consistent with the distribution of nAChRs in human brain, accumulated radioactivity was greatest in thalamus, intermediate in the midbrain, pons, cerebellum, and cortex; and least in white matter. As approximately 90% of the injected radioactivity was eliminated via the urine (biological half-life ca. 4 h), the urinary bladder wall received the highest radiation dose. The estimate of radiation dose equivalent to the urinary bladder wall (ca. 180 +/- 30 mSv/MBq or 0.7 rem/mCi with a 2.4 h void interval) suggests that multiple studies could be performed in a single subject. The results predict that quantitative PET imaging of nAChRs in human brain with 2-[18F]FA is feasible.