Characterization of [3H]Hemicholinium-3 Binding Sites in Human Brain Membranes: A Marker for Presynaptic Cholinergic Nerve Terminals

We report here on the binding properties of [3H]hemicholinium-3, a selective inhibitor of the high-affinity choline uptake process, to human brain membranes. Under the assay conditions described, the binding of [3H]hemicholinium-3 exhibited a dependency of physiological conditions on pH, temperature, and NaCl concentrations. Striatal binding proved to be specific, to a single site, saturable, and reversible, with an apparent KD of 10 nM and a Bmax of 82 fmol/mg of protein. [3H]Hemicholinium-3 specific binding exhibited a pharmacological profile and an ionic dependency suggestive of physiologically relevant interactions and comparable with those reported for the high-affinity choline uptake. Moreover, specific [3H]hemicholinium-3 binding exhibited an uneven regional distribution: striatum much greater than nucleus basalis greater than spinal cord much greater than midbrain = cerebellum greater than or equal to hippocampus greater than neocortex = anterior thalamus greater than posterior thalamus much much greater than white matter. This distribution closely corresponds to the reported activity of both enzymatic cholinergic presynaptic markers and high-affinity choline uptake in mammalian brain. There are no significant differences between these results and those previously found in the rat brain using this radioligand. Our results demonstrate, for the first time, the presence of [3H]hemicholinium-3 binding sites in human brain and strongly support the proposal that this radioligand binds to the carrier site mediating the high-affinity choline uptake process on cholinergic neurons. Thus, [3H]hemicholinium-3 binding may be used in postmortem human brain as a selective and quantifiable marker of the presynaptic cholinergic terminals.     

Are 5-HT receptors involved in the sprouting of serotoninergic terminals following neonatal 5,7-dihydroxytryptamine treatment in the rat?

The neonatal administration of 5,7-dihydroxytryptamine to rats (100 mg kg^?1 s.c. on the 1st and 2nd day after birth) resulted in marked reductions in serotoninergic presynaptic markers ([³H]-5-HT synaptosomal uptake, tryptophan hydroxylase activity and endogenous 5-HT content) in various forebrain areas, particularly the cerebral cortex and the hippocampus. In contrast, this treatment produced an increased outgrowth of serotoninergic terminals in the brain stem as judged by the significant increments of these presynaptic markers in this region. Both in the hippocampus and the brain stem, these 5,7-dihydroxytryptamine-induced changes in serotoninergic innervation were associated with a transient increase in 5-HT-sensitive adenylate cyclase activity. No significant alteration of the specific high affinity binding of [³H]-5-HT to synaptosomal membranes from various brain regions was detected in 5,7-dihydroxytryptamine-treated rats for at least the first postnatal month.The chronic blockade of 5-HT receptors by metergoline (5 mg kg^?1 day^?1 from day 3 to day 22 after birth) altered neither the changes in presynaptic markers nor the evolution of [³H]-5-HT high affinity binding in 5,7-dihydroxytryptamine-treated rats.These findings further illustrate that the high affinity binding sites for [³H]-5-HT do not correspond to postsynaptic 5-HT receptors coupled to adenylate cyclase in the rat brain. Apparently, 5-HT receptors play no role in the increased outgrowth of serotoninergic systems in the brain stem following neonatal 5,7-dihydroxy-tryptamine treatment.     

Altered Muscarinic and Nicotinic Receptor Densities in Cortical and Subcortical Brain Regions in Parkinson's Disease

Abstract: Muscarinic and nicotinic cholinergic receptors and choline acetyltransferase activity were studied in postmortem brain tissue from patients with histopathologically confirmed Parkinson's disease and matched control subjects. Using washed membrane homogenates from the frontal cortex, hippocampus, caudate nucleus, and putamen, saturation analysis of specific receptor binding was performed for the total number of muscarinic receptors with [³H]quinuclidinyl benzilate, for muscarinic M₁ receptors with [³H]pirenzepine, for muscarinic M₂ receptors with [³H]oxotremorine-M, and for nicotinic receptors with (–)-[³H]nicotine. In comparison with control tissues, choline acetyltransferase activity was reduced in the frontal cortex and hippocampus and unchanged in the caudate nucleus and putamen of parkinsonian patients. In Parkinson's disease the maximal binding site density for [³H]quinuclidinyl benzilate was increased in the frontal cortex and unaltered in the hippocampus, caudate nucleus, and putamen. Specific [³H]pirenzepine binding was increased in the frontal cortex, unaltered in the hippocampus, and decreased in the caudate nucleus and putamen. In parkinsonian patients B_max values for specific [³H]oxotremorine-M binding were reduced in the cortex and unchanged in the hippocampus and striatum compared with controls. Maximal (–)-[³H]nicotine binding was reduced in both the cortex and hippocampus and unaltered in both the caudate nucleus and putamen. Alterations of the equilibrium dissociation constant were not observed for any ligand in any of the brain areas examined. The present results suggest that both the innominatocortical and the septohippocampal cholinergic systems degenerate in Parkinson's disease. The reduction of cortical [³H]oxotremorine-M and (–)-[³H]nicotine binding is compatible with the concept that significant numbers of the binding sites labelled by these ligands are located on presynaptic cholinergic nerve terminals, whereas the increased [³H]pirenzepine binding in the cortex may reflect postsynaptic denervation supersensitivity.     

The effect of chronic L-DOPA administration on supersensitive pre- and postsynaptic dopaminergic receptors in rat brain

Chronic administration of haloperidol induced supersensitivity of the pre- and postsynaptic dopaminergic receptors in rat brain. The response of the presynaptic receptors was determined by an enhanced inhibitory effect of apomorphine on dopamine synthesis after gamma-butyrolactone injection. This change in the receptor function was detected both in the nigrostriatal and mesolimbic pathways. Haloperidol also increased the ³H-spiperone binding sites in striatal membranes, indicating supersensitivity of the postsynaptic receptors. Subsequent prolonged treatment with high doses of L-DOPA/carbidopa resulted in a decrease in ³H-spiperone binding sites, but had no effect on the supersensitive presynaptic receptors. It is suggested that tardive dyskinesia may be a state of both pre- and postsynaptic dopamine receptor supersensitivity and that chronic L-DOPA treatment may have a differential effect on these sites.     

Biochemical Assessment of Serotonergic and Cholinergic Dysfunction and Cerebral Atrophy in Alzheimer's Disease

Abstract: Markers of serotonin synapses in entire temporal lobe and frontal and temporal neocortex were examined for changes in Alzheimer's disease by use of both neurosurgical and autopsy samples. Uptake of [³H]sero-tonin, binding of [³H]imipramine, and content of indola-mines were all significantly reduced, indicating that serotonin nerve terminals are affected. Binding of [³H]serotonin was also reduced, whereas that of [³H]qui-nuclidinyl benzilate, [³H]muscimol, and [³H]dihydroal-prenolol were unaltered. When the Alzheimer's samples were subdivided according to age, the reduction in [³H]serotonin binding was a feature of only autopsy samples from younger patients. In contrast, presynaptic cholinergic activity was reduced in all groups of Alzheimer's samples, including neurosurgical specimens. Five markers, thought to reflect cerebral atrophy, cytoplasm, nerve cell membrane, and neuronal perikarya were measured in the entire temporal lobe. In Alzheimer's disease the reductions (mean 25%, range 20–35%) were thought to be too large to be due only to loss of structures associated with the presumed cholinergic perikarya in the basal forebrain and monoamine neurones in the brain stem.     

Pre- Versus Postsynaptic Localization of Benzodiazepine and β-Carboline Binding Sites

[3H]Flunitrazepam (FNP) and [3H]methyl beta-carboline-3-carboxylate (MCC) binding was examined in soluble and particulate fractions from membranes solubilized with Triton X-100 or in subfractions of synaptosomal membranes obtained by a physical separation technique. Results using both methods demonstrate that benzodiazepine and beta-carboline sites reside on both pre- and postsynaptic membranes. Further, subfractionation experiments indicate that the binding sites for both ligands are unequally distributed within the synapse and among brain regions. For example, in cerebral cortical presynaptic membranes there are twice as many FNP as MCC sites whereas in postsynaptic membranes this ratio is reversed. The number of FNP and MCC sites are equal in the presynaptic fraction from cerebellum. The postsynaptic membranes derived from cerebellum have three times the number of FNP compared to MCC sites. In hippocampus this ratio varies between 1.5 and 2.8 in each subfraction. These results support the idea that benzodiazepine and beta-carboline binding sites represent different recognition sites.     

Barbiturate competition for TRH receptors in mouse brain: neuromodulation of anesthesia

In vitro thyrotropin releasing hormone (TRH) radioligand binding assays were performed using purified presynaptic and postsynaptic membranes derived from various regions of mouse brain. These studies revealed the pattern of central distribution of specific TRH binding sites. The highest concentrations of both types of membrane receptors were localized in the limbic forebrain. The brain stem contained a high density of only presynaptic receptors, and the cerebral cortex contained a moderate-high level of only postsynaptic receptors. Barbiturate analogues effectively competed for all forebrain and brain stem, but not cortical, TRH receptors, thus implicating these specific receptors in the neuromodulation of barbiturate anesthesia. The results of in vivo radioligand binding assays for [3H] TRH disposition after central infusions concomitant with barbiturate vs. saline challenges further support this viewpoint.     

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl-β-carboline-3-carboxylate Binding Sites in Rat Brain Membranes

Abstract: This study was designed to analyze possible differences in the binding of [³H]flunitrazepam ([³H]FNZP) and [³H]ethyl - β - carboline - 3 - carboxylate ([³H]β-CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [³H]β-CCE was higher than for [³H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [³H]FNZP and [³H]β-CCE sites were equivalent; but later on, the β-carboline sites increased to a higher level. Noradrenergic denervation by 6-hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [³H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP-4 denervation. This change was only in B_max and not in K_D, In contrast, the [³H]β-CCE sites did not change with denervation. Neonatal injection of l - 2,4,5 - trihydroxyphenylalamine or DSP-4 produced in the adult a decrease in [³H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [³H]β-CCE did not change in the different brain regions. With 0.1% Triton X-100, applied to synaptosomal membranes, [³H]FNZP binding was reduced by 35%, while that of [³H]β-CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X-100, is that [³H]FNZP binds to pre- and postsynaptic receptors, while [³H]β-CCE binds mainly to postsynaptic benzodiazepine receptors.     

Biochemical Evidence of Selective Nerve Cell Changes in the Normal Ageing Human and Rat Brain

Abstract: Atrophy with ageing of human whole brain, entire temporal lobe, and caudate nucleus was assessed in autopsy specimens, by biochemical techniques. Only the caudate nucleus showed changes. Markers for several neurotransmitter systems were also examined for changes with age. In neocortex and temporal lobe of human brain, small decreases were detected in markers of cholinergic nerve terminals, whereas a large decrease (79%) occurred in the caudate nucleus. Findings were similar in striatum from 3–33-month-old rats. No change occurred in binding of [³H]quinuclidinyl benzilate by human samples. Markers of serotonergic terminals were also unchanged in human and rat brain. By contrast, binding of [³H]lysergic acid diethylamide and [³H]serotonin was decreased (32–81%) in human neocortex and temporal lobe, but not in caudate nucleus. A 43% loss of a marker of γ-aminobutyrate terminals occurred in human neocortex, while [³H]muscimol binding increased (179%). No changes were detected in markers of catecholamine synapses in temporal lobe or rat striatum. Hence, with human ageing there appears to be a loss of markers of γ-aminobutyrate neurones intrinsic to neocortex and acetylcholine cells intrinsic to the caudate nucleus, as well as a change in postsynaptic serotonin receptors in neocortex. These losses are accompanied by relative preservation of markers of ascending projections from basal forebrain and brain stem.     

Isolation of Hippocampal Synaptosomes on Percoll Gradients: Cholinergic Markers and Ligand Binding Sites

The S1 Percoll procedure, devised empirically for cortical tissue, provides highly purified, functionally viable synaptosomes on a four-step Percoll gradient. Here, for the first time, the procedure has been applied to rat hippocampus, and the gradient fractions have been analysed with respect to cholinergic markers and the synaptosomal index, lactate dehydrogenase. The presynaptic cholinergic markers choline acetyltransferase and [3H]choline uptake were most enriched in fraction 4. In contrast, acetylcholinesterase activity was broadly distributed across the gradient, consistent with the separation of synaptic plasma membranes (in fractions 1 and 2) from synaptosomes (in fractions 3 and 4). This is supported by the recovery of muscarinic binding sites labelled with [3H]quinuclidinylbenzilate in fractions 1 and 2. (-)-[3H]-Nicotine binding sites, however, were most enriched in fraction 4, consistent with their predominantly presynaptic localisation in the CNS. These results demonstrate the applicability of the S1 Percoll method to discrete brain regions for the recovery of homogeneous and viable synaptosome fractions. The separation of presynaptic terminals from post-synaptic membranes is a further advantage of this technique.     

“Specific” [3H]8-OH-DPAT binding to glass fiber filter paper: Implications for the analysis of serotonin binding site subtypes

The effect of buffer constituents on [³H]8-OH-DPAT binding to glass fiber filter paper was examined. Apparent “specific” [³H]8-OH-DPAT binding to glass fiber filter paper can be demonstrated when the radioligand binding assay is performed in the absence of 0.1% ascorbate. This artifactual “specific” binding is time dependent and appears to saturate. In addition, drug competition studies reveal complex interactions with [³H]8-OH-DPAT binding to glass fiber filter paper in the absence of ascorbate. Both 5-HT and chlorimipramine appear to “complete” for the sites labeled by [³H]8-OH-DPAT while both d-LSD and methysergide cause an increase in the [³H]8-OH-DPAT binding to glass fiber filter paper at micromolar concentrations. These data indicate that [³H]8-OH-DPAT binding to glass fiber filter paper may lead to misinterpretation of radioligand data obtained using brain homogenates in the absence of ascorbate.     

[3H]Dopamine Labeling of D3 Dopaminergic Sites in Human, Rat, and Calf Brain

Abstract: The binding of [³H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [³H]dopamine binding sites, with a B_max value of 185 fmol/mg protein, whereas rat and human striatum contained one-third this number of sites. The K_D values for [³H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin, and N-propylnorapomorphine) inhibited the binding of [³H]dopamine in all three species, at low concentrations, with IC₅₀ values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC₅₀ values of 200 to 40,000 nM). The [³H]dopamine binding sites were saturable, heat-labile, and detectable only in dopamine-rich brain regions; these sites differed from D₂ dopamine sites (labeled by [³H]butyrophenone neuroleptics), and from D_l dopamine sites (labeled by [³H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. We have, therefore, called these high-affinity [³H]dopamine binding sites D₃ sites. [³H]Apomorphine and [³H]ADTN also appeared to label D₃ sites. These ligands however, were less selective than [³H]dopamine, and labeled sites other than D₃ as well. Assay conditions were important in determining the parameters of [³H]dopamine binding. The optimum conditions for selective labeling of the D₃ dopaminergic sites, using [³H]dopamine, required the presence of EDTA and ascorbate.     

Discovery and characterization of [H]8-OH-DPAT binding to HeLaS3 cells

Some G protein-coupled receptors (GPCRs) have functional links to cancer biology, yet the manifestation of GPCRs in tumor types is little studied to date. Using a battery of radioligand binding assays, we sought to characterize GPCR recognition binding sites on HeLaS3 tumor cells. High levels of binding of the selective serotonin 5-HT_1A receptor agonist [³H]8-OH-DPAT were observed in these cells. Saturation and homologous competition experiments indicated that [³H]8-OH-DPAT bound different populations of high- and low-affinity sites. In competition experiments, several serotonergic compounds displaced [³H]8-OH-DPAT binding with low potency from its high-affinity binding sites, suggesting that low-affinity binding is the predominant mode of binding. A variety of drugs targeting different classes of receptors did not affect [³H]8-OH-DPAT binding. These observations may help elucidate the pathophysiological and functional relevance of 5-HT receptors in tumor cells and link GPCRs and tumorigenic mechanisms to pharmacological and chemotherapeutic paradigms.     

Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain

Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6-hydroxydopamine or 5, 7-dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6-hydroxydopamine- and 5,7-dihydroxytryptamine-lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.     

HT22 hippocampal neuronal cell line possesses functional cholinergic properties

AimsHippocampal cholinergic hypofunction is known to be involved in the cognitive deficits of Alzheimer's disease, but the detailed mechanisms remain to be elucidated. In order to establish an in vitro hippocampal cholinergic neuronal model for the relevant mechanistic studies, we have characterized a widely used hippocampal neuronal cell line, HT22, a sub-line derived from parent HT4 cells that were originally immortalized from primary mouse hippocampal neuronal culture.Main methodsWestern blot and immunocytochemistry were used to examine expression of cholinergic markers in HT22 cells. High potassium-evoked [³H]ACh release was used to evaluate the cholinergic functional properties of the cells.Key findingsWe found that HT22 cells express essential cholinergic markers, such as the high affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter, and muscarinic acetylcholine receptors. Exposure of HT22 cells to high potassium evoked [³H]ACh release in a dose-dependent manner. In addition, the [³H]ACh release was significantly potentiated when presynaptic autoreceptors were blocked.SignificanceOur results suggest that HT22 cells possess functional cholinergic properties, and can be used for an in vitro model for defining the mechanisms in cognitive deficits of Alzheimer's disease.     

[3H]Clonidine binding to rat hippocampal membranes

Alpha adrenergic receptor subtypes in rat hippocampal membranes were studied, using [³H]clonidine as the radioactive ligand. On the basis of competitive binding studies, using the selective antagonist-prazosin, WB-4101, and yohimbine, [³H] clonidine appeared to bind to a population of presynaptic sites that are pharmacologically similar to receptors previously classified as alpha₂. A computerized model that linearized and produced the best possible fit to the experimental data points indicated that [³H]clonidine binds to a single population of receptors possessing equal affinity for the ligand. Binding data also indicated that rat hippocampus contains significantly fewer [³H]clonidine binding sites than rat cortex.     

Mouse brain distribution of a carbon-11 labeled vesamicol derivative: presynaptic marker of cholinergic neurons

The regional mouse brain distribution of a new carbon-11 labeled derivative of vesamicol, [11C]-5-(N-methylamino)benzovesamicol [( 11C]MABV) is reported. Radiotracer concentrations in vivo are in the rank order of striatum greater than cortex greater than hippocampus greater than hypothalamus greater than cerebellum, consistent with reported distributions of other presynaptic cholinergic neuronal markers. In time course studies, striatum/cerebellum and cortex/cerebellum ratios for (-)-[11C]MABV continue to increase to values of 13 and 5, respectively, 75 min after i.v. injection of [11C]MABV. The specific binding in striatum and cortex is lowered by pretreatment with (+/-)-vesamicol, and shows stereoselectivity with lower uptake and lower ratios for the (+)-enantiomer. (-)-enantiomer. (-)-[11C]MABV is proposed as a positron-emitting radioligand for the in vivo study of presynaptic cholinergic neurons.     

High-Affinity Choline Transport Sites: Use of [3H]Hemicholinium-3 as a Quantitative Marker

Abstract: High-affinity choline transport (HAChT), the rate-limiting and regulatory step in acetylcholine (ACh) synthesis, is selectively localized to cholinergic neurons. Hemicholinium-3 (HC3), a potent and selective inhibitor of HAChT, has been used as a specific radioligand to quantify HAChT sites in membrane binding and autoradiographic studies. Because both HAChT velocity and [³H]HC3 binding change as in vivo activity of cholinergic neurons is altered, these markers are also useful measures of cholinergic neuronal activity. Evidence that [³H]HC3 is a specific ligand for HAChT sites on cholinergic terminals is reviewed. The ion requirements of HAChT and [³H]HC3 binding indicate that sodium and chloride are required for recognition of both choline and [³H]HC3. A common recognition site is also indicated by the close correspondence of the potency of HC3 and choline analogues for inhibiting both HAChT and [³H]HC3 binding. The parallel regional distributions of both markers in adult brain, during development and after specific lesions, all indicate specific cholinergic localization. The close association of HAChT and [³H]HC3 binding sites is also supported by parallel regulatory changes occurring after in vivo drug treatments and in vitro depolarization. Overall, the data indicate a close association between HAChT and [³H]HC3 binding and are consistent with the sites being identical. Methodologic considerations in using [³H]HC³ as a ligand and considerations in interpretation of results are also discussed.     

Bisquaternary Pyridinium Oximes as Presynaptic Agonists and Postsynaptic Antagonists of Muscarinic Receptors

A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1,1'-(4-hydroxyiminopyridinium)trimethylene (TMB-4) (I50 = 8 microM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) (I50 = 150 microM). As postsynaptic antagonists, the latter drugs are more potent (K1 = 1.3-3.3 microM) than TMB-4 (K1 = 50 microM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyperactivity of the cholinergic system.     

Nicotinic binding sites in cerebral cortex and hippocampus in alzheimer's dementia

Postmortem cerebral neocortical and hippocampal samples were taken from patients who died with dementia of the Alzheimer type (DAT) and individuals without diagnoses of neurological or psychiatric disease (control). Nicotinic binding was assayed with 20 nM [³H]acetylcholine ([³H]ACh) in the presence of atropine, or with 4 nM (-)-[³H]Nic). Binding of both ligands was lower in the following regions from DAT vs. control brains (P0.05): superior, middle and inferior temproal gyri, orbital frontal gyrus, middle frontal gyrus, pre- and postcentral gyri, inferior parietal lobule, and hippocampal endplate. Values of the correlation coefficient (r's) for binding of the nicotinic cholinergic ligands in these regions ranged from 0.70 to 0.93 (P's<0.05), suggesting that [³H]ACh and (-)-[³H]Nic labeled the same sites in human brain. There was no difference in nicotinic binding in the presubiculum, comparing DAT and control samples (P>0.05). Here too, correlations between binding of the two ligands were statistically significant in control and DAT groups (r's=0.92,P's<0.05). Nicotinic binding measured with [³H]ACh, but not (-)-[³H]Nic, was significantly lower in the H₂ (field of Rose) and H₁-subiculum areas of DAT samples compared to control. Correlations between binding of the two ligands in these regions ranged from 0.21 to 0.34 for the two groups (P's>0.05). The findings support a loss of neocortical and hippocampal nicotininc cholinergic binding sites in DAT. Further study is necessary to better characterize the regional losses of nicotinic binding in DAT and to resolve the differences in binding measured by [³H]ACh and (-)-[³H]Nic in the H₁-subiculum and H₂ (field of Rose) regions.