Autoradiographic analysis of D1 and D2 dopaminergic receptors in rat brain after paradoxical sleep deprivation

Previous work had shown that paradoxical sleep deprivation (PSD) results in potentiation of several apomorphine-induced behaviors, leading to the suggestion that PSD induces an upregulation of brain dopamine receptors. In this study, quantitative receptor autoradiography was used to verify whether PSD does, in fact, induce alterations in D₁ or D₂ receptor binding, and to investigate the regional brain specificity of such effects. After 96 h of PSD, [³H]SCH-23390 binding to D₁ receptors was examined in 30 different brain areas of 10 experimental and 10 cage control rats. [³H]Spiperone was used to label D₂ sites in adjacent tissue sections. Results revealed a 39% increase in [³H]SCH 23390 binding in the entorhinal cortex of PSD rats (p < 0.05), but no other changes in any of the remaining 29 brain areas examined. In contrast, [³H]spiperone binding was significantly elevated in the n. accumbens (+45%) and in all subrogions of the caudate-putamen (range: +13% to +23%). These results, thus, provide evidence that PSD increases D₂ but not D₁ receptor binding in brain. The present results also suggest that upregulated D₂ receptors can account for the previously reported changes in apomorphine-induced behaviors after PSD.     

Effects of NaCl and sultopride on striatal [H]spiperone binding in neonatal, adult and senescent rats

Effects of NaCl, (+)-and (−)-sultopride on striatal [³H]spiperone binding was investigated in 7-day, 70-day and 2-year-old rats. The amount of specific [³H]spiperone binding was the highest at 70 days and the value at adult stage was significantly (P < 0.001) higher than those at 7 days and 2 years. NaCl (100 mM) significantly increased [³H]spiperone binding in neonatal (P < 0.01), adult (P < 0.05) and senescent (P < 0.05) animals. Scatchard analysis showed that the Bmax of low-affinity [³H]spiperone binding was significantly elevated by 100 mM NaCl compared to the value in control of adult animals. More potent inhibition of (−)-sultopride for [³H]spiperone binding than that of the (+)-enantiomer at adult stage was also observed at neonatal and senescent stages. NaCl (100 mM) significantly enhanced inhibitory activities of (+)- and (−)-sultopride at every stage. It is suggested that stabilizing effect of Na⁺ on dopamine (DA) receptor complexes and increasing effect of Na⁺ on binding affinity of benzamide to DA₂ receptors keep functions through development and aging.     

Autoradiographic localization of D1 dopamine receptors in the rat brain with [H]SCH 23390

The regional distribution of D₁ dopamine (DA) receptors in the rat brain has been studied by quantitative autoradiography using the specific D₁ antagonist [³H]SCH 23390 as a ligand. The binding of [³H]SCH 23390 to striatal sections was saturable, stereospecific, reversible and of high affinity (K_d = 2.05nM); it occurred at single population of sites and possessed the pharmacological features of the D₁ DA receptor. The highest densities of [³H]SCH 23390 binding sites were found in the caudate-putamen, olfactory tubercle, nucleus accumbens and substantia nigra (especially in the pars compacta). High densities were also observed in the nucleus interstitialis striae terminalis, the anterior olfactory nucleus, the entopeduncular nucleus, the subthalamic nucleus, the claustrum and the amygdalohippocampal area. An intermediate labelling was found in the anteromedial and suprarhinal DA terminal fields of the cerebral cortex, the basolateral, medial and lateral amygdaloid nuclei, the endopiriform nucleus, the primary olfactory cortex, the globus pallidus, the superior colliculus (especially the superficial layer), the nucleus amygdaloideus corticalis and the dorsal hippocampus (molecular layer of the CA₁ and dentate gyrus). In the anteromedial and suprarhinal cortices, [³H]SCH 23390 binding was more concentrated in layers V and VI. Moderate levels of [³H]SCH 23390 were found in the thalamus, hypothalamus, the habenula, the ventral tegmental area, the posterior cingulate and entorhinal cortices, the supragenual dopamine terminal system and the cerebellum (molecular layer). This regional distribution of [³H]SCH 23390 closely correlated (except for the cerebellum) with the reported distribution of dopaminergic terminals. The topographical distribution of [³H]SCH 23390 has also been studied in detail in striatal subregions. The density of D₁ receptors was much greater in the ventrolateral sector and medial margin of the striatum than in the ventromedial and dorsolateral sectors. A rostrocaudal decrease in the densities of D₁ sites was also found along the rostrocaudal axis of the caudate-putamen. These lateral to medial and anteroposterior gradients overlapped with the density of the dopaminergic afferents.     

[H]Nemonapride Binding in Human Caudate and Putamen

The binding of [³H]nemonapride to human postmortem caudate and putamen tissue was autoradiographically investigated using several antipsychotic drugs. Saturation experiments revealed a single population of binding sites (dissociation constant (K_D) 0.38 ± 0.01 nM, and total binding capacity (B_MAX) 55 fmol/TE). Prototypic dopamine (DA) receptors antagonists displaced [³H]nemonapride in a monophasic manner. The order of displacement potency was expected for DA D₂-like receptors: spiperone > (+)butaclamol ≥ chlorpromazine > (−)sulpiride > ketanserin. Displacement with serotonergic antagonists suggests that in human caudate and putamen tissue [³H]nemonapride may have a very low affinity serotonergic component. However, [³H]nemonapride displays a high affinity and selectivity for DA D₂-like receptors and should make it a preferred compound for tritium-based autoradiography.     

SDZ GLC 756, a novel octahydrobenzo[g]quinoline derivative exerts opposing effects on dopamine D1 and D2 receptors

Summary SDZ GLC-756, a novel octahydrobenzo[g]quinoline derivative, is equipotent in displacing [³H]SCH23390 from dopamine D₁ receptors and [³H]205–501 from dopamine D₂ receptor binding sites. It blocks dopamine sensitive adenylate cyclase with the same potency as SCH23390, indicating antagonist properties at dopamine D₁ receptors. On the other hand, SDZ GLC 756 inhibits electrically evoked acetylcholine release from rat striatal slices with the same potency as the selective dopamine D₂ receptor agonist bromocriptine. This effect is blocked by spiperone suggesting that it is mediated by dopamine D₂ receptor activation. The opposing action of SDZ GLC 756 on dopamine D₁ and D₂ receptors is also evident in vivo. SDZ GLC 756, like SCH23390, blocks apomorphine-induced rearing in mice. On the other hand, it inhibits prolactin secretion and produces circling in unilateral 6-OHDA-lesioned rats, which is compatible with stimulant properties at dopamine D₂ receptors. This drug might be a new tool to study linkage between dopamine D₁ and D₂ receptors.     

Autoradiographic comparison of D1-specific binding of [H]SCH39166 and SCH23390 in the primate cerebral cortex

Quantitative autoradiography was used to compare the binding of the novel dopamine D₁ receptor antagonist, [³H]SCH39166, with that of the widely used radioligand, [³H]SCH23390 (in the presence of ritanserin), in the primate cerebral cortex. Specific binding of both radioligands, determined using SCH23390 or cis-flupentixol as displacing agents, had very similar densities and distributions throughout the cortex. However, the specific binding of [³H]SCH39166 obtained with SCH39166 as a blank was significantly higher than that obtained using SCH23390 or cis-flupentixol as displacing agents in some layers of motor, somatosensory and occipital cortices. In addition, the non-specific binding of [³H]SCH39156 obtained in the presence of an excess of SCH23390 of cis-flupentixol displayed a complex laminar pattern very different from that of the specific binding. These observations suggest that [³H]SCH39166 may have a high affinity to more than the D₁ receptor subtype bound by SCH23390 or cis-flupentixol. Also, these additional sites are likely to be different from 5-HT₂ or 5-HT_1C receptors since the latter sites were not displaced by 1 μM SCH23390.     

The novel anticonvulsant MK-801: a potent and specific ligand of the brain phencyclidine/gd-receptor

MK-801 (5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine maleate) is a novel anticonvulsant agent reported to antagonize certain N-methyl-spd-aspartate (NMDA)-mediated effects non-competitively. The question arises of the mechanism underlying the anti-NMDA and anticonvulsant effects of MK-801. In the present study MK-801 is shown to be an extremely potent inhibitor of the binding of N-[³H](1-[2-thienyl]cyclohexy)piperidine) ([s3H]TCP to brain phencyclidine (PCP)/δ-receptors. It is IC₅₎ value of 3.8 ± 0.8 nM in this assay ranks it as the most potent known ligand of brain PCP/δ-receptors. Addition of MK-801 altered the apparent K_d but not the apparent B_max values for [³H]TCP binding, indicating a competitive interaction. The specificity of action of MK-801 is supported by the finding that MK-801 strongly inhibited the binding of (+)-N-[³H]allylnormetazocine ((+)-[³H]SKF 10.047) to the PCP/δ-receptor but its effect on (+)-[³H]SKF 10.047 binding to the non-PCP, haloperidol-sensitive δ-binding site was weaker by several orders of magnitude. Furthermore, MK-801 exerts PCP-like antagonistic effects upon NMDA-induced [³H]norepinephrine release. These findings support the concept that the anticonvulsant and anti-NMDA effects of MK-801 result from its being the most potent known ligand of PCP/δ-receptors.     

Alteration of nicotinic cholinergic agonist binding sites in hippocampus after fimbria transection

Nicotinic cholinergic agonist binding sites were studied in rat hippocampus by the binding of [³H]acetylcholine in the presence of 1.5 μM atropine sulfate. Following transection of the fimbria/fornix there was a 49% increase in the binding of [³H]acetylcholine reflecting an increase in the affinity of the receptor binding site from K_d = 18.82 ± 3.6 nM in control animals to K_d = 9.06 ± 1.2 nM in experimental tissue. Chronic administration of the agonist nicotine (4 mg/kg/day) by osmotic minipumps produced an increased in the binding of 10 nM [³H]acetylcholine after 14 days (49% increase over control) and after 28 days (141% increase over controls). These data are consistent with the suggestion that [³H]acetylcholine labels a nicotinic cholinergic receptor in rat brain. Further they support the notion that some of the termination sites of the septal-cholinergic projection to the hippocampus are nicotinic.     

Dopamine D-2 receptors in rat caudate-putamen: the lateral to medial gradient does not correspond to dopaminergic innervation

The topography of dopamine D-2 receptors within the rat caudate-putamen (CPU) was characterized by measuring [³H]spiroperidol binding to membranes from dissected regions of the CPU and to thin sections of rat forebrain using quantitative autoradiography. Specific binding, defined using 1 μM (+)-butaclamol, was considerably higher in the lateral CPU than the medial CPU, without any obvious gradient in the dorsoventral axis. The difference in B_max between the lateral and medial CPU was similar whether determined by digital subtraction autoradiography of coronal forebrain sections or by [³H]spiroperidol binding to membrane homogenates derived from CPU subregions. No regional differences were observed in the affinity of [³H]spiroperidol for D-2 sites. There were no lateral-to-medial differences in the dopaminergic innervation of the CPU, as determined by the measurement of the levels of dopamine, homovanillic acid and dihydroxyphenylacetic acid in subregions of the CPU using high pressure liquid chromatography. Thus, regional variations in the density of D-2 receptors within the CPU do not correspond to the degree of dopaminergic innervation. The functional importance of the organization of this dopamine receptor system is discussed in relation to the lateral-to-medial organization of the CPU.     

Characterization and quantitative autoradiography of [H]tryptamine binding sites in rat brain

[³H]tryptamine binds with high affinity (K_d = 9.1nM, B_max= 54fmol/mg wet wt.) to tissue sections of rat brain. The binding occurs rapidly and is reversible. Low concentrations of the β-carbolines harmaline (IC₅₀ = 25nM) and tetrahydronorharman (tetrahydro-β-carboline), IC₅₀ = 50nM) inhibit [³H]tryptamine binding. Serotonin (5-HT, IC₅₀ = 2600nM) as well as the 5-HT receptor antagonists methysergide and metergoline displace [³H]tryptamine at much higher concentrations from brain slices. The distribution of [³H]tryptamine binding sites in section of rat brain has been analyzed by quantitative autoradiography. The highest density of binding sites is found in the nucleus (n.) interpreduncularis, a slightly lower one in the locus coeruleus. Moderately labelled are the n. accumbens septi, n. septi lateralis, n. medalis habenulae, n. tractus olfactorii lateralis, the central region of the amydgala, n. caudatsu/putamen, n. reuniens and the hippocampal formation. A low density of binding sites is detected in the cerebral cortex and the subiculum. Even less binding sites are found in the n. dorsalis raphe and the substantia nigra. The pattern of distribution of [³H]tryptamine binding sites differs from that of [³H]5-HT(5-HT₁), [³H]ketanserin (5-HT₂) as well as [³H]imipramine binding sites. These data suggest unique tryptamine binding sites.     

Distribution of α-adrenergic, β-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat

Catecholamine receptor binding sites were measured in discrete hypothalamic nuclei or regions as well as in certain extrahypothalamic areas of the adult male rat. For each assay, discrete areas were microdissected from frozen tissue sections and pooled from several animals. Specific high affinity binding sites were assessed at fixed ligand concentrations for [³H]p-aminoclonidine (PAC) and [³H](2-C 2′,6′-(CH₃O)₂ phenoxyethylamino)-methylbenzodioxan (WB-4101) for α-adrenergic receptor sites, for [³H]dihydroalprenolol (DHA) for β-adrenergic receptor sites, and for [³H]2-amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphtalene (ADTN) and [³H]spiroperidol in the presence of cinanserin for dopaminergic receptor sites.Regional variations in [³H]WB-4101 binding were relatively small in magnitude, with most hypothalamic and extrahypothalamic areas possessing between 60 and 90% of the binding in frontal cortex. [³H]PAC binding showed a wider range of binding density across brain areas than did [³H]WB-4101, but, in general, variations in [³H]PAC binding paralleled those in [³H]WB-4101 binding. In hypothalamus, binding was characterized as being predominantly to α₁-receptors in the of [³H]WB-4101 and to α₂-receptors in the case of [³H]PAC. The medial hypothalamic areas exhibited a somewhat higher density of these α-adrenergic sites than did the lateral hypothalamus (perifornical hypothalamus and medial forebrain bundle). Also, the ratio of [³H]PAC to [³H]WB-4101 binding differed in different hypothalamic areas, ranging from 1.5:1 to 4:1. The median eminence was exceptional in that it contained appreciable [³H]PAC but no significant [³H]WB-4101 binding sites at the radioligand concentrations tested. Binding of [³H]DHA to β-adrenergic receptors varied over approximately a 3-fold range in the different hypothalamic areas, with binding highest in the medial forebrain bundle and the medial preoptic area, and lowest in the periventricular, dorsomedial and posterior hypothalamic nuclei, the median eminence and the zona incerta. The ratio of β-adrenergic to α-adrenergic binding sites was generally lower in the medial than in the lateral hypothalamic areas and higher in the extrahypothalamic areas examined than in the hypothalamus. With regard to [³H]spiroperidol and [³H]ADTN binding to dopaminergic sites, the striatum, nucleus accumbens and olfactory tubercle showed a greater density of [³H]spiroperidol than of [³H]ADTN sites, in contrast to the hypothalamus where [³H]ADTN binding was more predominant. Within the hypothalamus, [³H]ADTN binding was relatively uniform, while [³H]spiroperidol binding was quite high in four hypothalamic areas (lateral perifornical area, medial forebrain bundle, paraventricular and dorsomedial nuclei), intermediate in the median eminence and arcuate nucleus, and low or not detectable in all other hypothalamic areas.     

Binding of (+)PHNO and other D2-dopamine agonists to D1-dopamine receptors labelled by [3H]SCH 23390

Summary The affinity of (+)PHNO for the D₁-dopamine receptor subtype in striatal tissue was determined in a radioligand binding assay using the D₁-selective compound, [³H]SCH 23390. The IC₅₀ for (+)PHNO was 22M, a value considerably higher than obtained for reference agents (lisuride, apomorphine, pergolide, [–]EOE and bromocriptine). The low potency of (+)PHNO at the D₁-receptor subtype contrasts with the compound's high potency at the D₂-receptor subtype, as reported previously. (+)PHNO can, therefore, be classified as a very selective D₂-receptor agonist.     

Age-dependent changes in the expression of dopamine receptor subtypes in human peripheral blood lymphocytes

The pharmacological profile and the density of dopamine D₃ and D₅ receptor subtypes expressed by human peripheral blood lymphocytes of subjects of different ages (ranging from 20 to 75 years) were assessed using radioligand binding techniques. Dopamine D₃ receptor was assayed with [³H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([³H]7-OH-DPAT) as a ligand. Dopamine D₅ receptor was assayed using [³H][R]-(+)-(-chloro-2,3,4,5,tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate) ([³H]SCH 23390) as a ligand. The affinity and the pharmacological profile of [³H]7-OH-DPAT and [³H]SCH 23390 at dopamine D₃ and D₅ receptor, respectively, were similar in subjects of different ages. The density of dopamine D₃ receptor binding sites was slightly decreased in subjects of 30–39 years in comparison with younger individuals. A remarkable loss of dopamine D₃ receptor was then found between 40 and 49 years of age in comparison with younger subjects. A further slight decrease was noticeable between 50 and 59 years of age. The number of [³H]7-OH-DPAT binding sites was then stabilized after 60 years of age. The density of dopamine D₅ receptor binding sites did not show age-dependent changes. The above findings indicate the occurrence of a decline in the density of lymphocyte dopamine D₃ but not D₅ receptor between adult and mature subjects. The possibility that dopamine D₃ receptor assay in peripheral blood lymphocytes may represent a tool for investigating dopamine receptor function in aging and age-related neurological disorders is discussed.     

Striatal D1- and D2-dopamine receptor sites are separately detectable in vivo

We have characterized in particulate fractions of normal rat striatum the in vivo binding kinetics, binding affinity, and pharmacological profiles of [3H]SCH 23390, a ligand selective for the D1-subtype of dopamine (DA) receptor, and compared these to [3H]spiperone, a ligand classically associated with the D2 DA receptor subtype. The pharmacological specificity of each ligand's in vivo binding is very similar to binding to striatal homogenates in vitro. While similar maximum numbers (Bmax) of striatal binding sites exist in vivo compared to in vitro for both ligands, binding affinities in vivo for both ligands are reduced 125- to 200-fold compared to in vitro. In vivo binding of [3H]SCH 23390 to striatum is not increased by dopamine denervation produced by 6-hydroxydopamine lesions of the nigrostriatal pathway. In vivo binding of [3H]SCH 23390 and [3H]spiperone to striatum is not significantly reduced by increased synaptic concentration of dopamine following D-amphetamine administration. 125I-SCH 23982, the iodinated analogue of SCH 23390, localizes very highly to dopaminergic forebrain areas following i.v. administration. External imaging of mammalian and human brain D1-receptors is potentially feasible with this ligand.     

The interaction between β- and α2-adrenoceptors in cerebral cortical membranes: Isoproterenol-induced increase in [H]clonidine binding in rats

The pretreatment of rat cerebral cortical membranes with 10 or 100 μM isoproterenol at 37°C for 40 min caused a significant elevation of the B_max value of [³H]clonidine binding but pretreatment at 4°C did not affect the value. The isoproterenol-induced increase in the B_max value of the binding was higher in 50 mM Tris-HCl buffer (pH 7.7) than in Krebs-Ringer solution. In 50 mM Tris-HCl buffer (pH 7.7), treatment with isoproterenol reduced the B_max value of [³H]dihydroalprenolol binding but neitherK_d nor B_max of [³H]WB 4101 binding was affected by this treatment. Fitty μM propranolol or 100 μM GTP produced a significant reduction in isoproterenol-induced elevation of the B_max value of [³H]clonidine binding. In contrast, 100 μM cyclic AMP did not affect the control binding and 0.1 or 1 mM theophylline did not affect the isoproterenol-induced elevation of the binding. The only B_max value in high affinity binding of [³H]clonidine was increased by isoproterenol.It is suggested that isoproterenol increases the density of α₂-adrenoceptors in a temperature-dependent manner. The direct interaction between β- and α₂-receptor molecules and/or their indirect interaction, mediated by GTP regulatory proteins, would exist in the cerebral cortical membranes of rats.     

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [H]idazoxan and [H]rauwolscine

The regional distribution of [³H]idazoxan and [³H]rauwolscine was studied autoradiographically in human brain. [³H]Idazoxan binds with high affinity to α₂ adrenoceptors as well as to non-adrenergic sites (NAIBS). [³H]Rauwolscine, besides binding to α₂ adrenoceptors, also binds to 5-HT_1A receptors. Both radioligands labelled the same population of α₂ adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of α₂ adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no α₂ adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable α₂ adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT_1A receptors labelled by [³H]rauwolscine is in agreement with previous studies using [³H]8-OH-DPAT.     

Differential labeling of dopamine and sigma sites by [H]nemonapride and [H]raclopride in postmortem human brains

The difference between the binding of [³H]nemonapride and [³H]raclopride has been used to quantify dopamine D₄ receptors in postmortem schizophrenic brain studies. Recent work, however, has suggested that at least part of the differential between [³H]nemonapride and [³H]raclopride binding may represent σ rather than D₄ receptor sites. We applied the nemonapride-raclopride subtraction method to postmortem, non-schizophrenic human striatum to examine the variation in dopaminergic receptor binding labeled by these ligands. Variation in σ receptor binding labeled by [³H]nemonapride was studied in frontal cortex, striatum and cerebellum. Specific binding was defined by sulpiride (dopamine receptor ligand), PPAP (σ receptor ligand) and haloperidol (mixed dopaminergic/σ agent), respectively. Haloperidol defined a combination of sites, which were approximately the sum of the dopaminergic and σ components defined by sulpiride and PPAP, respectively. Significant inter-individual variation in the amount of specific binding for dopaminergic and σ receptor sites was observed. However, no significant nor consistent observation of striatal dopamine D₄ receptors or D₄-like binding sites was observed in the striatum even though two independent sets of tissues, with different dissections were used. The inconsistencies in some previous postmortem studies appear to be at least partially explained by the inclusion of both σ and dopaminergic components in [³H]nemonapride binding and the inherent high inter-individual variability of the different components.     

Dopamine receptors in the anterior lobe of the human pituitary gland: autoradiographic localization

We examined the distribution of dopamine and serotonin receptors in human pituitary gland by light microscopic autoradiography using [³H]spiperone as a binding ligand. Clusters of specific binding sites for [³H]spiperone were observed in the anterior pituitary. This clustering is similar to the clustering of lactotrophs observed in immunohistochemical studies. No specific binding sites for [³H]spiperone were present in the posterior lobe of the pituitary gland. We found no evidence for the presence of serotonin-2 receptors (i.e. cinanserin-displaceable [³H]spiperone binding) in human pituitary.     

Modulation of dopamine receptor supersensitivity by chronic insulin: implication in schizophrenia

Haloperidol-induced increases in the number of dopamine receptors, as measured by [³H]spiperone binding to striatal membranes, do not occur in rats repeatedly treated with insulin in doses eliciting pronounced hypoglycemia. Given alone, however, insulin has no effect on [³H]spiperone binding in normal rats. These findings demonstrate a modulating effect of insulin on brain dopamine receptor sensitization. This effect might be relevant to the mechanism of insulin coma therapy in schizophrenia and is consistent with and supports the dopaminergic hypothesis of this disorder.     

Autoradiographic localization of muscarinic agonist binding sites in the rat central nervous system with (+)-cis-[H]methyldioxolane

(+)-cis-[³H]Methyldioxolane ((+)-[³H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[³H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[³H]CD states and binding sites for the muscarinic antagonists [³H]pirenzepine (r = −0.79) and (-)-[³H]quinuclidinyl benzilate (r = −0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.