Localization of opioid receptor antagonist [3H]-LY255582 binding sites in mouse brain: comparison with the distribution of mu, delta and kappa binding sites.

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.

Characterization of neuropeptide Y Y1-like and Y2-like receptor subtypes in the mouse brain.

To characterize receptor subtypes in the mouse, we performed autoradiographic localization and pharmacological characterization studies using the selective radiolabeled agonists, [(125)I]-Leu(31), Pro(34)-PYY and [(125)I]-PYY 3-36. The pharmacology of [(125)I]-Leu(31), Pro(34)-PYY and [(125)I]-PYY 3-36 binding to mouse brain homogenates were consistent with Y1-like and Y2-like receptors, respectively. Using receptor autoradiography, high Y1-like binding was observed in the islands of Calleja and dentate gyrus. [(125)I]-PYY 3-36 binding was highest in the hippocampus, lateral septum, stria terminalis of the thalamus, and compacta and lateralis of the substantia nigra. In addition, there are differences in receptor distribution in mouse brain compared to other species that may translate into different functional roles for the NPY receptors within each species.

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes.

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.

Pharmacological characterization of (125)I-1229U91 binding to Y1 and Y4 neuropeptide Y/Peptide YY receptors.

1229U91 (GW1229 or GR231118) [lle,Glu,Pro,Dpr,Tyr, Arg,Leu,Arg, Tyr-NH(2))2 cyclic (2,4'),(2'4)-diamide] has been reported by several research groups to be a potent antagonist at the Y1 neuropeptide Y (NPY) receptor subtype. However, 1229U91 also displaces (125)I-peptide YY (PYY) with high affinity from the Y4 subtype. Previously, we reported that 1229U91 had full agonist properties for the Y4 receptor. To characterize the pharmacological properties of 1229U91 directly, we had it radioiodinated with the chloromine-T method. (125)I-1229U91 bound to cell lines expressing the human Y1 and Y4 receptors with high affinity. The K(d) and B(max) for (125)I-1229U91 binding to Y1 were 14.9 pM and 1458 fmol/mg protein, respectively. The Y4 receptor bound (125)I-1229U91 with a K(d) of 12.5 pM and a B(max) of 1442 fmol/mg protein. When competing (125)I-1229U91 binding from Y1 and Y4 receptors, a similar rank order of potency was observed: 1229U91 > [Leu(31),Pro(34)]-NPY >/= [Leu(31),Pro(34)]-PYY > PYY >/= NPY > NPY(2-36) > PYY(3-36). Pancreatic polypeptide (PP) potently displaced (125)I-1229U91 from the Y4 receptor, but displayed little affinity for Y1. In autoradiographic studies with rat brain sections, (125)I-1229U91 bound with a distribution similar to that reported for the Y1 receptor when localized with (125)I-[Leu(31),Pro(34)]-PYY. Brain regions exhibiting binding sites for (125)I-PP were not detected with this radioligand. Those include the interpeduncular nucleus and the periventricular nucleus of the hypothalamus. Furthermore, (125)I-labeled rat PP was not displaced from these areas with 10 nM 1229U91. Thus, (125)I-1229U91 is a high affinity Y1 and Y4 radioligand and binds with a distribution in the rat brain consistent with the localization of the Y1 receptor.

Structure-activity relationship of a series of diaminoalkyl substituted benzimidazole as neuropeptide Y Y1 receptor antagonists.

A series of benzimidazoles (4) was synthesized and evaluated in vitro as potent and selective NPY Y1 receptor antagonists. Substitution of the piperidine nitrogen of 4 with appropriate R groups resulted in compounds with more than 80-fold higher affinity at the Y receptor compared to the parent compound 5 (R = H). The most potent benzimidazole in this series was 21 (Ki = 0.052 nM).

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites.

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu31, Pro34]NPY, and NPY(3-36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.

Synthesis and evaluation of a series of novel 2-[(4-chlorophenoxy)methyl]benzimidazoles as selective neuropeptide Y Y1 receptor antagonists.

A series of novel benzimidazoles (BI) derived from the indole 2 was synthesized and evaluated as selective neuropeptide Y (NPY) Y1 receptor antagonists with the aim of developing antiobesity drugs. In our SAR approach, the (4-chlorophenoxy)methyl group at C-2 was kept constant and a series of BIs substituted with various piperidinylalkyl groups at N-1 was synthesized to identify the optimal spacing and orientation of the piperidine ring nitrogen relative to the benzimidazole. The 3-(3-piperidinyl)propyl in 33 was found to maximize affinity for the Y1 receptor. Because of the critical importance of Arg33 and Arg35 of NPY binding to the Y1 receptor, the incorporation of an additional aminoalkyl functionality to the structure of 33 was explored. Methyl substitution was used to probe where substitution on the aromatic ring was best tolerated. In this fashion, the C-4 was chosen for the substitution of the second aminoalkyl functionality. Synthesis of such compounds with a phenoxy tether using the 4-hydroxybenzimidazole 11 was pursued because of their relative ease of synthesis. Functionalization of the hydroxy group of 45 with a series of piperidinylalkyl groups provided the dibasic benzimidazoles 55-62. Among them, BI 56 demonstrated a Ki of 0.0017 microM, which was 400-fold more potent than 33. To evaluate if there was a stereoselective effect on affinity for these BIs, the four constituent stereoisomers (69-72) of the BI 60 were prepared using the S- and R-isomers of bromide 17. Antagonist activity of these BIs was confirmed by measuring the ability of selected compounds to reverse NPY-induced forskolin-stimulated cyclic AMP. The high selectivity of several BI antagonists for the Y1 versus Y2, Y4, and Y5 receptors was also shown.

Differential distribution of neuropeptide Y Y1 and Y2 receptors in rat and guinea-pig brains.

The distribution of neuropeptide Y Y1 and Y2 receptors has been extensively studied in the rat using selective masking of [125I]peptide YY binding by Y1- and Y2-selective peptides. In the present study, we characterized the binding of the subtype-selective radioligands [125I](Leu31-Pro34)-peptide YY and [125I]peptide YY3-36 to rat and guinea-pig brains and identified differences in Y1 and Y2 distribution. [125I](Leu31Pro34)-peptide YY and [125I]peptide YY3-36 bound to single sites in rat and guinea-pig brains with pharmacologies consistent with the Y1 and Y2 receptors, respectively. Autoradiographic studies were conducted using adjacent sections from rat and guinea-pig brains. Compared to the rat, the distribution of both Y1 and Y2 receptors was markedly different in the guinea-pig. For example, a high level of Y1 binding was detected within the thalamus of the rat while, in the guinea-pig, very little Y1 binding was observed in this region. Y1 binding was very low in the rat hippocampus, while the guinea-pig hippocampus contained high levels of Y1 binding. High levels of both Y1 and Y2 binding were observed in the guinea-pig cerebellum while, in the rat, only low levels of Y2 binding were visible. Therefore, the guinea-pig brain exhibits dramatic differences in the pattern of Y1 and Y2 receptors when compared to the rat. It is likely that Y1 and Y2 receptors perform different roles in the central nervous system of guinea-pigs and rats.

[125I]Leu31, Pro34-PYY is a high affinity radioligand for rat PP1/Y4 and Y1 receptors: evidence for heterogeneity in pancreatic polypeptide receptors.

Cloned receptors for the PP-fold peptides are subdivided into Y1, Y2, PP1/Y4, Y5 and Y6. NPY and PYY have similar affinity for Y1, Y2, Y5 and Y6 receptors while PP has highest affinity for PP1. Pro34-substituted analogs of NPY and PYY have selectivity for Y1 and Y1-like receptors over Y2 receptors. In the present study, we found the putative Y1-selective radioligand, [125I]Leu31, Pro34-PYY, also binds with high affinity to the rat PP1 receptor in cell lines expressing the receptor. However, in rat brain sections, [125I]Leu31, Pro34-PYY does not appear to bind to the interpeduncular nucleus, a brain region containing a high density of [125I]-bPP binding sites. Therefore, it appears there is additional heterogeneity in receptors recognizing PP.

Pharmacological characterization of neuropeptide Y-(2-36) binding to neuropeptide Y Y1 and Y2 receptors.

Neuropeptide Y-(2-36) has been reported by several research groups to be a more potent orexigenic agent than intact neuropeptide Y. Therefore, it has been proposed that a novel 'Y1 variant' may modulate ingestive behavior. To define the receptor subtype involved in neuropeptide Y-stimulated feeding behavior, we evaluated the binding properties of neuropeptide Y-(2-36) and [125I]neuropeptide Y-(2-36) in established neuropeptide Y1 and Y2 containing cell lines and tissues. Neuropeptide Y-(2-36) displaced [125I]peptide YY binding to SK-N-MC cells (neuropeptide Y Y1 receptors) with a Ki of 3.69 nmol and SK-N-BE(2) cells (neuropeptide Y Y2 receptors) with a Ki of 3.08 nmol. Neuropeptide Y-(2-36) also displaced [125I]peptide YY binding to rat cerebral cortex, hippocampus and olfactory bulb with similar affinities. To examine the brain distribution of [125I]peptide YY, [125I]neuropeptide Y and [125I]neuropeptide Y-(2-36), adjacent sections were labeled and the binding sites detected by autoradiography. A similar distribution of binding was observed for each radioligand in all regions examined. Therefore, neuropeptide Y-(2-36) binds non-selectively to neuropeptide Y Y1 and neuropeptide Y Y2 receptors, but with lower affinity than neuropeptide Y and peptide YY. The increased potency and selectivity seen with neuropeptide Y-(2-36) in feeding studies cannot be explained on the basis of a unique in vitro pharmacology.

The neuropeptide Y Y1 receptor selective radioligand, [125I][Leu31,Pro34]peptide YY, is also a high affinity radioligand for human pancreatic polypeptide 1 receptors.

A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings from pharmacology and molecular biology studies. Neuropeptide Y and peptide YY have similar affinity for neuropeptide Y Y1 and neuropeptide Y Y2 while pancreatic polypeptide has highest affinity for pancreatic polypeptide 1. Pro34-substituted analogs of neuropeptide Y and peptide YY have selectivity for neuropeptide Y Y1 over neuropeptide Y Y2 receptors. In the present study, we found that one such 'neuropeptide Y Y1-selective' radioligand, [125I][Leu31,Pro34]peptide YY, also binds with high affinity to the pancreatic polypeptide 1 receptor. Therefore, caution needs to be exercised when using Pro34-analogs to define the neuropeptide Y Y1 receptor in vivo and using tissue preparations.

Expression cloning of a human brain neuropeptide Y Y2 receptor.

The 36-amino acid peptide, neuropeptide Y (NPY), is a member of a peptide family that includes the endocrine peptides, peptide YY (PYY), and pancreatic polypeptide (PP). NPY receptors have been broadly subdivided into postsynaptic Y1 receptors and presynaptic Y2 receptors based on the preference of Pro34-substituted analogues for the Y1 receptors and carboxyl-terminal fragments for the Y2. A Y1 receptor has been cloned, and this receptor appears to mediate several effects of NPY, including vasoconstriction and anxiolysis in animal models. We report the cloning of a human brain Y2 receptor from a human brain library. Pools of clones were transiently expressed in COS-1 cells, and 125I-PYY binding pools were identified by autoradiography. After a single positive pool was detected in the original screening, a single clone was isolated by four rounds of sequential enrichment. The clone encoded a 381-amino acid protein of the heptahelix (seven TM) type. Amino acid identity of this receptor with the Y1 receptor was 31% overall with 40% identity in the TM regions. Comparison with the human PP1 receptor indicated 33% overall amino acid identity with 42% identity in the TM regions. Pharmacologically, the receptor exhibited high affinity for NPY, PYY, and carboxyl-terminal fragments of NPY and PYY. In addition, Pro34-substituted analogues had very low affinity. With the use of Northern blot analysis, high levels of Y2 mRNA were detected in a variety of brain regions with little expression in peripheral tissues. Thus, the receptor protein has the pharmacological properties and distribution of the human Y2 receptor.

Unexpected high density of neuropeptide Y Y1 receptors in the guinea pig spleen.

Receptors for neuropeptide Y (NPY) and peptide YY (PYY) have been extensively characterized in the brain. Less is known about NPY receptor subtypes in the spleen, though it is well established that NPY produces vascular contraction in this tissue. In the present study, we found an unusually high density of Y1 receptors in the guinea pig spleen. These receptors are localized to the red pulp and exhibit a pharmacology that is consistent with the Y1 receptor. On the other hand, only very low densities for Y2 receptors were observed. Therefore, the guinea pig spleen may be a ideal tissue for further study of the role of Y1 receptors in cardiovascular and immune function.

[3H]-quinelorane binds to D2 and D3 dopamine receptors in the rat brain.

Quinelorane is a BCD partial ergoline with potent dopaminergic effects in vitro and in vivo. Partial ergoline compounds of this series consist of the B-, C- and D-rings of the four ring ergoline skeleton. Many of the pharmacological effects of quinelorane are believed to be due to stimulation of the D2 subtype of the dopamine receptor. Recently, a D3 dopamine receptor was identified that is insensitive to guanine nucleotides and exhibits an unusual distribution in the brain. When this receptor is expressed in Chinese hamster ovary cells, quinelorane has higher affinity for the D3 receptor than the D2 receptor. To further define the pharmacology of quinelorane, we have synthesized [3H]-quinelorane and examined its binding to sections of rat brain in vitro. [3H]-quinelorane bound with high affinity (KD = 1.8 nM) and exhibited very low nonspecific binding. D2 selective antagonists, such as (+)butaclamol and spiperone, were potent inhibitors of binding while the D1 antagonist SCH23390 was significantly less potent. A majority of the binding was inhibited in a concentration-dependent manner by guanylyl-imidodiphosphate with a maximal inhibition at concentrations of 1 microM and greater. Autoradiographic studies were performed in the presence and absence of 10 microM Gpp(NH)p. Binding in D2 containing regions, such as the caudate-putamen, was completely inhibited by guanylyl-imidodiphosphate although binding in D3 containing areas, such as the islands of Calleja, was unaffected. Therefore, [3H]-quinelorane is an excellent agonist radioligand for the localization of D2 and D3 receptors in rat brain.

Comparison of (R)-[3H]tomoxetine and (R/S)-[3H]nisoxetine binding in rat brain.

(R)-[3H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of (R)-[3H]tomoxetine with those of (R/S)-[3H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas (R)-[3H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of (R)-[3H]tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where (R/S)-[3H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and (R)-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 nM (R)-nisoxetine to mask the binding of (R)-[3H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [3H]citalopram. Therefore, (R)-[3H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.

Autoradiographic comparison of [125I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain.

Although the density and distribution of 5-HT2A (5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized 125I-lysergic acid diethylamide ([125I]LSD) to quantify and compare 5-HT2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 microM) and sulpiride (1 microM) were used to displace [125I]LSD binding from 5-HT2A and D2 binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.

Localization of neuropeptide Y immunoreactivity and [125I]peptide YY binding sites in the human pituitary.

High levels of neuropeptide Y (NPY) are found in the hypothalamus, median eminence, pituitary portal blood, and the pituitary in a number of species. Neuropeptide Y may influence the synthesis and secretion of a variety of hormones by interacting with specific receptors in the hypothalamus and/or the pituitary. To further define the function of NPY in the pituitary, we have examined the distribution of NPY immunoreactivity and NPY receptors in sections of human pituitary using immunohistochemical and autoradiographic techniques. Neuropeptide Y-immunoreactive varicose axons were seen throughout the neural lobe. A moderate number of NPY-immunoreactive cells were found in the anterior lobe. A very high level of [125I]PYY binding was seen in the neural lobe with low levels in the anterior lobe. The binding in the neural lobe was inhibited by NPY(13-36) at a Ki of 5.3 nM and [Leu31-Pro34]NPY at a Ki of 390 nM, indicating the receptor was the Y2 subtype. Therefore, neuronally released NPY may modulate human neural lobe function through a Y2 receptor.

Synthesis and evaluation of [125I]-(S)-iodozacopride, a high affinity radioligand for 5HT3 receptors.

We have developed a high specific activity radioiodinated ligand for the biochemical evaluation and autoradiographic localization of 5HT3 receptors in the brain. [125I]-(S)-iodozacopride was synthesized by radioiodination of deschloro-(S)-zacopride using chloramine-T, and the product was purified by HPLC. The equilibrium kinetics and pharmacology of the binding of this radioligand were studied in homogenates of rat cerebral cortex, while the distribution of binding was examined by quantitative autoradiography. [125I]-(S)-iodozacopride bound to a single, saturable, specific binding site (Kd = 192 +/- 9 pM, Bmax = 1.2 +/- 0.2 fmol/mg protein). The binding had the pharmacological properties of a 5HT3 receptor, being potently inhibited by a variety of 5HT3 agonists and antagonists including (S)-zacopride (Ki = 0.032 nM), Quipazine (Ki = 0.45 nM), LY278584 (Ki = 0.5 nM), (1-m-chlorophenyl)-biguanide (Ki = 0.6 nM) and ICS 205-930 (Ki = 1.0 nM). Autoradiographic studies were undertaken by incubating sections with 400 pM [125I]-(S)-iodozacopride and exposing them to film for 3-7 days to obtain suitable autoradiograms. Specific binding of [125I]-(S)-iodozacopride was found at various amounts in a variety of brain regions. The highest levels of binding were found in the brainstem, principally the nucleus of the solitary tract with somewhat lower levels in the area postrema, substantia gelatinosa of the trigeminal nucleus and dorsal motor nucleus of the vagus. In the rat forebrain, moderate levels of specific binding were found in the glomerular layer of the olfactory bulb, anterior olfactory nucleus and various subnuclei of the amygdala. Lower levels of binding were seen in the superficial laminae of the parietal cerebral cortex and diffusely distributed throughout the hippocampal formation. In conclusion, [125I]-(S)-iodozacopride binds to a receptor site with the pharmacological properties and distribution that is consistent with the 5HT3 receptor. [125I]-(S)-iodozacopride represents a significant improvement in autoradiographic studies of the 5HT3 receptor by reducing the required exposure time for producing autoradiograms from the 3-6 months required for [3H]-labeled ligands to 3-7 days.

Localization of rat brain binding sites for [3H]tomoxetine, an enantiomerically pure ligand for norepinephrine reuptake sites.

The distribution of binding sites for the potent inhibitor of norepinephrine (NE) reuptake, [3H]tomoxetine, was examined in rat brain using quantitative autoradiography. Scatchard analysis of [3H]tomoxetine-binding to slide-mounted sections of rat forebrain indicated that the ligand bound to two sites, a high-affinity site with a Kd of 0.29 nM and a lower-affinity site with a Kd of 16 nM. Pharmacological characterization of this high-affinity site was consistent with labelling a NE-uptake site in brain. Autoradiographic localization of the binding sites for [3H]tomoxetine was performed at a ligand concentration of 1 nM representing the distribution of high-affinity sites. The radioligand bound with a distribution of binding sites that was consistent with the known distribution of NE-containing neurons. The highest levels of binding were seen in regions, such as the locus coeruleus, bed nucleus of the stria terminalis, anterior ventral nucleus of the thalamus and the paraventricular nucleus of the hypothalamus. Low levels were seen in regions such as the caudate-putamen, ventral tegmental area and zona reticulata of the substantia nigra, where NE-containing neurons have been reported to be low. Binding to all these sites was inhibited by 1 microM desipramine which produced autoradiograms with a uniform nonspecific binding. These results indicate that low concentrations of [3H]tomoxetine can be used to localize and characterize NE-binding sites. Further study will be necessary to determine the nature of the low-affinity binding site.