3H]GBR-12935 binding to dopamine uptake sites in the human brain

The binding of the selective dopamine uptake inhibitor [3H]GBR-12935 to post-mortem human brain membranes was studied. Competition experiments indicated the presence of multiple binding sites, but when a binding fraction that could be discriminated by either 0.3 microM mazindol or 1 mM dopamine was regarded as specific binding, a single-site binding model was obtained. The [3H]GBR-12935 binding was of protein nature since it was abolished after protease treatment and the binding appeared to label the dopamine uptake site. This was supported by the findings that dopamine uptake inhibitors inhibited the binding with high affinity (Ki 30-130 nM), whereas substances active at dopamine D1, D2 or autoreceptor sites revealed much lower affinities (Ki greater than 10 microM or inactive). Moreover, dopamine was the neurotransmitter with the highest affinity for the [3H]GBR-12935 binding site (Ki 30 microM). The dopaminergic nature of the [3H]GBR-12935 binding was further indicated by its regional distribution, which largely corresponds the known distribution of the dopamine system in the rat brain. The highest binding densities were obtained in the caudate nucleus and putamen (Bmax 1500-2000 fmol/mg protein), followed by the olfactory tubercle (Bmax 900 fmol/mg protein) and the substantia nigra (Bmax 300 fmol/mg protein). The apparent binding affinity (Kd) was the same in all brain regions (Kd 1-1.5 nM). Detectable specific [3H]GBR-12935 binding was obtained also in the globus pallidus, amygdala and cortices of orbital/rectus and cingulate gyri. Drug inhibition studies with the addition of low concentrations of dopamine and mazindol produced only alterations in the apparent Kd values, suggesting a competitive inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity [3H]paroxetine binding to serotonin uptake sites in human brain tissue

[3H]Paroxetine binding to human brain tissue was characterized. Competition studies in the putamen and frontal cortex revealed single-site binding models for binding sensitive to 5-hydroxytryptamine (5-HT) (Ki 1-3 microM) and citalopram (Ki 0.6 nM), which displaced the same amount of binding. However, desipramine, norzimeldine and fluoxetine displaced additional binding (10-20%) and these competitors fitted two-site binding models with high affinity components in the nanomolar range and low affinity components in the micromolar range. The high affinity components approximated the 5-HT- and citalopram-sensitive binding fraction. Most of the [3H]paroxetine binding sites were protease-sensitive, but the low-affinity (microM) sites appeared to be protease-resistant. Based on these findings, only the [3H]paroxetine binding representing the fraction sensitive to 30 microM 5-HT (or e.g. 0.3 microM norzimeldine), was regarded as specific binding. This binding fraction was saturable with an apparent binding affinity (Kd) of 0.03-0.05 nM throughout the brain. The highest binding densities were obtained in the hypothalamus and substantia nigra (Bmax 500 fmol/mg protein). The basal ganglia reached intermediate densities (Bmax 200 fmol/mg protein), whereas cortical areas had low Bmax values (less than 100 fmol/mg protein). The lowest B max value was noted in cerebellar cortex (30 fmol/mg protein). The [3H]paroxetine binding was competitively inhibited by low concentrations of 5-HT, imipramine and norzimeldine, suggesting that the substrate recognition site for 5-HT uptake was labeled. Compounds active at dopaminergic, noradrenergic, histaminergic, 5-HT1, 5-HT2 and cholinergic muscarinic sites did not affect the binding at 100 microM concentrations. It is concluded that [3H]paroxetine is a marker for the 5-HT uptake site in the human brain, provided that an adequate pharmacological definition of specific binding is performed.     

Evidence for multiple [3H]prazosin binding sites in canine brain membranes

Two classes of alpha 1 adrenoceptors were identified in canine brain and liver using conventional radioligand binding methods. Scatchard plots of specific [3H]prazosin binding to brain and liver membranes prepared from 100-150-day-old Doberman pinscher dogs were consistently curvilinear and best fit a two-site binding model (frontal cortex, Kd1 = 57.7 +/- 10.0 pM, Bmax1 = 64.6 +/- 17.1 fmol/mg protein, Kd2 = 1.5 +/- 0.5 nM, Bmax2 = 159 +/- 37.6 fmol/mg protein; liver, Kd1 = 82.6 +/- 36 pM, Bmax1 = 7.0 +/- 5.1 fmol/mg protein, Kd2 = 0.8 +/- 0.2 nM, Bmax2 = 62.1 +/- 8.7 fmol/mg protein). Kinetically derived affinity constants from association and dissociation experiments agreed with those obtained by Scatchard analyses of equilibrium binding data. Binding sites were saturable, heat labile, bound ligand reversibly, and appeared to be appropriately distributed in relation to endogenous catecholamine. [3H]Prazosin also bound with high affinity to two classes of binding site in porcine and bovine brain membrane but [3H]prazosin binding in monkey and rat brain was best described by a single-site binding model. Affinities obtained were in between values obtained for high and low affinity Kds in the other species. Competitions for [3H]prazosin binding sites in canine frontal cortex were conducted with the following antagonists: WB-4101, corynanthine, phentolamine, benoxathian, phenoxybenzamine, chlorethylclonidine, thymoxamine, prazosin, yohimbine and agonists: methoxamine, (-)-norepinephrine, and clonidine. All ligands but prazosin, norepinephrine and clonidine competed for specific [3H]prazosin binding in a statistically significant biphasic manner. Benoxathian and WB-4101 displayed the highest affinities (benoxathian: Ki1 = 0.26 nM, WB-4101: Ki1 = 0.20 nM) and selectivity (high affinity/low affinity: benoxathian = 1640, WB-4101 = 13204) for the high affinity [3H]prazosin binding site; chlorethylclonidine had highest affinity (Ki2 = 91 nM) and selectivity (low affinity/high affinity = 405) for the lower affinity [3H]prazosin binding site. As defined, the two sites were similar to the alpha 1a and alpha 1b recently described in the rat and rabbit. A noticeable difference was that the subtypes described in dog brain had a 30-fold difference in affinity for prazosin.     

Properties of dopamine agonist and antagonist binding sites in mammalian retina

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.     

Binding of [3H]p-aminoclonidine to two sites, alpha 2-adrenoceptors and imidazoline binding sites: distribution of imidazoline binding sites in rat brain

Binding sites labeled by [3H]p-aminoclonidine [( 3H]PAC) were investigated by the competitive analysis with imidazoline and non-imidazoline derivatives. Phenylethylamine derivatives displaced only the part of specific sites for [3H]PAC, which was considered as alpha 2-adrenoceptor, whereas imidazoline derivatives, such as clonidine and tolazoline, competed for a further specific binding of [3H]PAC to the non-adrenergic sites, in addition to the alpha 2-adrenoceptor. Because the non-adrenergic sites were specific for the imidazoline structure, they were termed imidazoline sites. The imidazoline sites were not distributed uniformly among rat brain regions. In striatum, hippocampus and medulla oblongata, they occupied 39.6, 33.0 and 36.5% of the specific binding of [3H]PAC, respectively. Saturation isotherms revealed that Kd and Bmax of imidazoline sites for [3H]PAC were 3.09 +/- 0.59 nM, 27.4 +/- 1.7 fmol/mg protein and 2.23 +/- 0.29 nM, 21.0 +/- 1.5 fmol/mg protein in striatum and hippocampus, respectively. Because imidazoline binding sites also displayed weak affinities for imidazole compounds, such as histamine and cimetidine, the imidazoline site may be a subtype of histamine H2-receptor.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

5-Hydroxytryptamine-sensitive [3H]imipramine binding of protein nature in the human brain. I. Characteristics

[3H]Imipramine binding sites were characterized in the human brain by investigating the sensitivity to protease treatment, dependency on NaCl and the effects of drug inhibition. The binding was found to consist of a protease sensitive and a protease resistant fraction. These two fractions could be discriminated by 5-hydroxytryptamine (5-HT) but not desipramine. The [3H]imipramine binding discriminated by 5-HT was found to be sodium dependent. The 5-HT-sensitive [3H]imipramine binding displayed a regional variability with Bmax values ranging from 50 to 100 fmol/mg protein in neocortical areas to 400-500 fmol/mg protein in the substantia nigra and hypothalamus. The Kd values for 5-HT-sensitive [3H]imipramine binding were 1-2 nM throughout the brain. Additional [3H]imipramine binding insensitive to 5-HT, but displaceable by desipramine showed little regional variation, with the binding capacity in the hypothalamus approximating that found in cortical areas. This binding fraction was of low affinity, was not dependent on the presence of NaCl and was insensitive to protease treatment. Drug inhibition studies revealed that the addition of low concentrations of 5-HT or norzimeldine to 5-HT-sensitive [3H]imipramine binding sites produced changes in affinity, consistent with a competitive interaction. It is suggested that the 5-HT-sensitive [3H]imipramine binding may represent the substrate recognition site for 5-HT uptake in the human brain.     

Characterization and quantitative autoradiography of [3H]tryptamine binding sites in rat brain

[3H]Tryptamine binds with high affinity (Kd = 9.1 nM, Bmax = 54 fmol/mg wet wt.) to tissue sections of rat brain. The binding occurs rapidly and is reversible. Low concentrations of the beta-carbolines harmaline (IC50 = 25 nM) and tetrahydronorharman (tetrahydro-beta-carboline, IC50 = 50 nM) inhibit [3H]tryptamine binding. Serotonin (5-HT, IC50 = 2600 nM) 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 sections of rat brain has been analyzed by quantitative autoradiography. The highest density of binding sites is found in the nucleus (n.) interpeduncularis, a slightly lower one in the locus coeruleus. Moderately labelled are the n. accumbens septi, n. septi lateralis, n. medialis habenulae, n. tractus olfactorii lateralis, the central region of the amygdala, n. caudatus/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.     

Alterations in [3H]spiperone binding in human caudate nucleus, substantia nigra and frontal cortex in the Shy-Drager syndrome and Parkinson's disease

[3H]Spiperone binding was investigated in the caudate nucleus, substantia nigra (s. nigra) and frontal cortex of control subjects and of patients with Parkinson's disease and the Shy-Drager syndrome. Binding sites for [3H]spiperone were interpreted as dopamine receptors in caudate and s. nigra, and as 5-hydroxytryptamine (5-HT) receptors in frontal cortex. Scatchard analysis showed that the Bmax (maximal number of binding sites) in caudate was similar in the 3 groups, whereas in s. nigra the Bmax was reduced by approximately 60% in both Parkinsons disease and Shy-Drager syndrome. The dissociation constant (Kd) for [3H]spiperone binding in s. nigra was similar in the 3 groups. In caudate nucleus, the Kd was similar in control and Parkinson groups; however, there was a significant increase in the dissociation constant in the caudate nucleus from cases of Shy-Drager syndrome. No differences in binding characteristics were observed in the frontal cortex. These results are taken to reflect a loss of dopamine receptor sites in the s. nigra in both Parkinson's disease and Shy-Drager syndrome, and a reduced affinity of dopamine receptor sites in the caudate nucleus in Shy-Drager syndrome.     

Regional and subcellular localization in human brain of [3H]paroxetine binding, a marker of serotonin uptake sites

The characteristics of the binding of [3H]paroxetine, a selective serotonin (5-HT) uptake blocker, were investigated in human brain. The Kd value was 0.23 +/- 0.07 nM, and the Bmax value was 190 +/- 39 fmol/mg protein in the putamen. The capacity of various antidepressive drugs to inhibit [3H]paroxetine-specific binding in human brain was well correlated with their capacity to inhibit [3H]5-HT uptake in rat brain. The highest concentrations of [3H]paroxetine-specific binding sites were found in the substantia nigra, hypothalamus, and hippocampus. Lower values were obtained in the basal ganglia and the thalamus. The specific binding was very low in cerebral and cerebellar cortices. The regional distribution of [3H]paroxetine binding sites differs from that of [3H]ketanserin binding to S2 serotonin receptors. The subcellular distribution of the [3H]paroxetine-specific binding sites obtained by differential centrifugation revealed a synaptosomal enrichment in the frontal cortex and striatum, whereas an enrichment in the microsomal fraction was found in striatum. The results show that [3H]paroxetine is a ligand of choice to label the 5-HT uptake molecular complex in human brain.     

Characterization of benzodiazepine receptors in the bovine pineal gland: evidence for the presence of an atypical binding site

Bovine and rat pineal benzodiazepine receptors were characterized using ligands with high affinities for either 'central-type' (CBR) or 'peripheral-type' (PBR) benzodiazepine receptors. The characteristics (Bmax = 83 +/- 10 fmol/mg protein, Kd = 3.88 +/- 0.46 nM) of benzodiazepine receptors in bovine pineal membranes measured with [3H]flunitrazepam (using flunitrazepam to define non-specific binding) were consistent with previously reported values. However, if non-specific binding was defined using Ro 15-1788 (a selective CBR ligand), the Bmax and Kd of [3H]flunitrazepam decreased 51 and 58%, respectively. In addition, when using PK 11195 to determine non-specific binding, the Bmax of [3H]flunitrazepam binding to bovine pineal decreased further (approximately 80%, Kd decreased approximately 39%). Together, these observations strongly suggested the presence of PBR in the bovine pineal. Bovine pineal PBR characterized with [3H]PK 11195 revealed a high density (relative to CBR) of high affinity binding sites (Kd = 1.08 +/- 0.30, Bmax = 776 +/- 33.0 fmol/mg protein). In contrast, when [3H]Ro 5-4864 (1-20 nM) was used to define PBR, no binding was detectable. These observations are in sharp contrast to the rat pineal gland, in which both [3H]Ro 5-4864 and [3H]PK 11195 bind to a large number of PBR with high affinity (Kd approximately equal to 1.9 nM, Bmax approximately equal to 26 pmol/mg protein). Bovine pineal PBR were further characterized with compounds structurally related to either Ro 5-4864 or PK 11195.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of striatal and nigral tachykinin receptors in the rat

The effects of lesioning mesostriatal dopamine projections or striatal neurons on tachykinin binding in the basal ganglia were assessed in the rat. 6-Hydroxydopamine lesions of the medial forebrain bundle destroyed striatal dopamine terminals as assessed by [³H]mazindol autoradiography, but did not significantly affect the binding of NK-1 ([³H][Sar⁹, Met(O₂)¹¹]substance P) or NK-3 ([³H]senktide) tachykinin ligands in the striatum. 6-Hydroxydopamine lesions significantly reduced NK-3 binding in the substantia nigra pars compacta, but not the ventral tegmental area. In contrast, striatal quinolinic acid lesions reduced both NK-1 and NK-3 binding in the striatum, but failed to affect NK-3 binding in the substantia nigra. These findings suggest that both NK-1 and NK-3 receptors within the striatum are predominantly post-synaptic with respect to dopamine neurons, whereas nigral NK-3 receptors are located on dopaminergic neurons.     

VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes.

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studied in primary cultures, indicates the presence of a single class of high affinity binding sites with a Kd of 3.3 nM and a Bmax of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis. Secretin, despite its lack of interaction with sites labeled by M-[125I]VIP, stimulates glycogenolysis with an EC50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a Kd of 41.3 nM and a Bmax of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with Kd of 1.4 and 30.3 nM, and Bmax of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a Kd of 4.9 nM and a Bmax of 316 fmol/mg protein, and a low affinity class with a Kd of 42.8 nM and a Bmax of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[125I]VIP with an EC50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[125I]VIP exclusively for the neuronal class of binding sites.     

Adenosine uptake and [3H]nitrobenzylthioinosine binding in developing rat brain

The ontogenesis of adenosine transport sites as labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined using radioligand binding and membrane preparations from whole brain and 4 brain regions of rats between the postnatal ages of one day through to adulthood. In whole brain, cerebral cortex and cerebellum, [3H]NBI binding was two-fold higher in 6-day-old than in 50-day-old rats. In contrast, [3H]NBI binding was higher in adults than in one-day-old rats by 4-fold in hypothalamus and 8-fold in superior colliculus. In cortex and hypothalamus, the levels of [3H]NBI binding in newborn and adult rats were reflected by changes in Bmax and not Kd values. As a measure of the utility of [3H]NBI as a probe for identifying functional adenosine transport sites, we examined [3H]NBI binding to and [3H]adenosine accumulation by intact brain cells prepared from adult and newborn rats. For [3H]NBI binding to brain cells from adult rats, the values of Kd were 0.092 nM and of Bmax were 274 fmol/mg protein. For newborns, slightly higher Kd and Bmax values were observed; 0.2 nM and 395 fmol/mg protein, respectively. [3H]Adenosine accumulation was higher in brain cells from one-day-old than from adult rat brains. Kinetically this uptake was best described by a two-component model: the Vmax values for the high- and low-affinity uptake, and the Km value for the high-affinity component in one-day-old rats were greater than in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and differential regulation of GABAA and benzodiazepine receptors in rat neocortex.

We have characterized the gamma-aminobutyric acid-A (GABAA) and benzodiazepine (BZ) receptors in in vitro living slices of adult rat neocortex using [3H]SR95531, a GABAA antagonist, and [3H]flunitrazepam (FNZ), a BZ ligand. [3H]SR95531 labelled a single population of GABAA receptors with a Bmax of 1030.7 fmol/mg protein and a Kd of 43.5 nM. [3H]FNZ also labelled a single binding site with a Bmax of 4239 fmol/mg protein and a Kd of 22 nM. The GABAA receptor labelled using [3H]SR95531 could be down-regulated by 2 h preincubations in GABA and the GABAA agonist muscimol (8% and 11%, respectively). Increases in cellular electrical activity induced by a combination of veratridine and glutamate led to an average increase in GABAA receptor number of 58%. The BZ binding site labelled with [3H]FNZ was down-regulated by clonazepam (-55%), increased by GABA (+17%), but not altered by changes in electrical activity. The present results demonstrate the rapid differential regulation of a ligand-gated receptor by agonist stimulation or increases in bioelectric activity. Such regulation may provide clues to the nature of the modifications which occur following changes in cellular activity in the cortex.     

The distribution of M1 and M2 muscarinic acetylcholine receptor subtypes in the developing cat visual cortex

The binding site characteristics and ontogenesis of [3H]pirenzepine ([3H]PZ) (M1 receptor) and [3H]oxotremorine-M ([3H]OXO-M) (M2 receptor) binding sites were investigated in the cat visual cortex. Scatchard analysis of [3H]PZ binding in adult cat visual cortex revealed a single site with a Kd of 17.3 nm and a Bmax of 352.45 fmol/mg protein. [3H]OXO-M also bound to a single site with a Kd of 7.1 nM and a Bmax of 256.39 fmol/mg protein. Receptor autoradiography revealed that [3H]PZ binding sites were present only in telencephalic structures while [3H]OXO-M sites were distributed heterogeneously throughout the brain. [3H]PZ binding sites in adult visual cortex were present in the superficial and deep cortical layers with the densest labeling in layer I and a distinct band in layer V. [3H]OXO-M sites also avoided the middle cortical layers, but were most prominent in layers V and VI with less pronounced binding in layers I and II. Deafferentation of extrinsic inputs to the visual cortex did not reduce [3H]PZ nor [3H]OZO-M binding, but neuron-specific excitotoxic lesions of visual cortex abolished both populations of binding sites. This indicates that both populations of binding sites are located on cells intrinsic to the cortex. In early postnatal life, both [3H]PZ and [3H]OXO-M binding sites were localized to intermediate cortical layers. Following this, the laminar distribution of both populations redistributed; each with its own idiosyncratic profile. By postnatal day 49, [3H]PZ binding sites redistributed into the superficial and deep layers, the pattern of adult animals, while [3H]OXO-M sites maintained a pattern similar to younger animals, with substantial binding persisting in layer IV. As late as postnatal day 70, well after [3H]PZ binding sites had achieved their mature laminar pattern, [3H]OXO-M binding sites in visual cortex had not achieved their characteristic adult pattern. In addition, the normal laminar redistribution of both [3H]PZ and [3H]OXO-M binding sites during postnatal development of the cat visual cortex was prevented by eliminating cortical afferents in early postnatal life. This indicates that muscarinic receptor rearrangement in development is dependent upon cortical input or output.     

High- and low-affinity [3H]desipramine-binding sites in human postmortem brain tissue

The binding of [3H]desipramine to human brain tissue was characterized. Competition studies in the frontal cortex and hypothalamus revealed a single-site binding model for noradrenaline (Ki 120-190 microM). The noradrenaline uptake inhibitors nisoxetine, nortriptyline and desipramine fitted two-site binding models and these compounds exhibited 10-80 times lower Ki values than the serotonin uptake inhibitor citalopram. The high-affinity component of the nisoxetine-sensitive [3H]desipramine binding (Ki 50-110 nM) approximated the binding sensitive to noradrenaline. This binding fraction was defined as that sensitive to 1 microM nisoxetine and showed a maximum binding capacity (Bmax) of 380 +/- 80 fmol/mg protein and an apparent Kd of 5.1 (4.5-5.7) nM in the hypothalamus. The binding was also investigated in 25 additional brain regions without finding detectable amounts of binding. However, when the specific binding was defined as that sensitive to 100 microM nisoxetine, low-affinity binding where Bmax and Kd were not possible to determine was obtained in all brain regions investigated. It is concluded that [3H]desipramine binding to human brain tissue represents multiple binding sites. Only when regarding binding sensitive to noradrenaline and to the high-affinity component of noradrenaline uptake inhibitors is the binding saturable and of high affinity. It is possible that this site represents the uptake site for noradrenaline.     

Picomolar affinity of 125I-SCH 23982 for D1 receptors in brain demonstrated with digital subtraction autoradiography

Iodinated SCH 23390, 125I-SCH 23982 (DuPont-NEN), was examined using quantitative autoradiography for its potency, selectivity, and anatomical and neuronal localization of binding to the dopamine D1 receptor in rat brain sections. 125I-SCH 23982 bound to D1 sites in the basal ganglia with very high affinity (Kd values of 55-125 pM), specificity (70-85% of binding was displaced by 5 microM cis-flupenthixol), and in a saturable manner (Bmax values of 65-176 fmol/mg protein). Specific 125I-SCH 23982 binding was displaced by the selective D1 antagonists SCH 23390 (IC50 = 90 pM) and cis-flupenthixol (IC50 = 200 pM) and the D1 agonist SKF 38393 (IC50 = 110 nM) but not by D2-selective ligands (I-sulpiride, LY 171555) or the S2 antagonist cinanserin. Compared with 3H-SCH 23390, the 5- to 10-fold greater affinity for the D1 site and 50-fold greater specific radioactivity of 125I-SCH 23982 makes it an excellent radioligand for labeling the D1 receptor. The concentrations of D1 sites were greatest in the medial substantia nigra and exceeded by over 50% the concentration of D1 sites in the lateral substantia nigra, caudoputamen, nucleus accumbens, olfactory tubercle, and entopeduncular nucleus. Lower concentrations of D1 sites were present in the internal capsule, dorsomedial frontal cortex, claustrum, and layer 6 of the neocortex. D1 sites were absent in the ventral tegmental area. Intrastriatal injections of the axon-sparing neurotoxin, quinolinic acid, depleted by 87% and by 46-58% the concentrations of displaceable D1 sites in the ipsilateral caudoputamen and medial and central pars reticulata of the substantia nigra, respectively. No D1 sites were lost in the lateral substantia nigra. Destruction of up to 94% of the mesostriatal dopaminergic projection with 6-hydroxydopamine did not reduce D1 binding nor, with one exception, increase striatal or nigral D1 receptor concentrations. 125I-SCH 23982 selectively labels D1 binding sites on striatonigral neurons with picomolar affinity, and these neurons contain the majority of D1 sites in rat brain.     

The localization of receptor binding sites in the substantia nigra and striatum of the rat

Neurotransmitter receptor binding of 5 ligands was examined in the striatum, substantia nigra (SN) and frontal cortex of rats which had received either unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway (NSP) or unilateral kainic acid lesions of the striatum. 6-OHDA lesions of the NSP significantly reduced [3H]dihydroalprenolol ([3H]DHA) and [3H]naloxone ([3H]Nal) binding by 31% and 28% respectively, in the denervated striatum compared to the contralateral side. Scatchard analysis revealed that the alteration in [3H]DHA binding was not due to a change in the affinity of the beta-adrenergic receptor for [3H]DHA. In marked contrast to these changes in the striatum, destruction of the NSP resulted in a significant increase in [3H]DHA and [3H]Nal binding by 44% and 26%, respectively, in the frontal cortex of the lesioned compared to the control side. 6-OHDA lesions in the NSP did not alter striatal receptor binding for [3H]quinuclidinyl benzilate ([3H]QNB), [3H]muscimol ([3H]Mus) or [3H]flunitrazepam ([3H]Flu). Similarily, intrastriatal kainic acid injections did not alter striatal receptor binding for [3H]Nal, [3H]Flu or [3H]Mus. Of the various receptor densities measured in the SN after the above lesions the only alteration observed was a 43% increase in [3H]Flu binding following 6-OHDA lesions of the NSP. Scatchard analysis indicated no change in the affinity of the benzodiazepine receptor for [3H]Flu. 6-OHDA lesions of the NSP did not alter [13H]QNB or [3H]Nal binding in the SN. Striatal kainic acid lesions did not alter nigral [3H]QNB or [3H]Flu binding. The results are discussed in terms of neurotransmitter localization and plasticity within the striatum, SN and frontal cortex.     

Functional recovery of supersensitive dopamine receptors after intrastriatal grafts of fetal substantia nigra

Interruption of the ascending dopamine neurons of the nigrostriatal pathway, by 6-hydroxydopamine (6-OHDA) lesion in rats, produced a significant loss of the dopamine transport complexes labeled with the phencyclidine derivative [3H]BTCP. This loss of dopamine innervation in the striatum was present at least 12 to 14 months after lesioning and was functionally manifested by ipsilateral rotation of the animals in response to amphetamine. In these same animals, in comparison to controls, there was a significant increase in the number (Bmax) of [3H]SCH 23390-labeled D-1 receptors in the striatum (36.7%) and the substantia nigra (35.1%) and a 54.4% increase in the number (Bmax) of [3H]sulpiride-labeled striatal D-2 receptors without an apparent change in affinity (Kd). Ten to twelve months after the transplantation of homologous fetal substantia nigra into the denervated striatum, there was a significant decrease in amphetamine-induced turning behavior. In these animals, there was an ingrowth of dopamine nerve terminals in the striatum as demonstrated by a return of [3H]BTCP binding. Accompanying this reinnervation was the normalization of D-1 and D-2 receptors to control values in the striatum as well as the return of D-1 receptors to prelesion densities in the substantia nigra. In a subgroup of transplanted rats, amphetamine continued to induce ipsilateral turning. In these animals both D-1 and D-2 receptors remained supersensitive. These results support the hypothesis that the functional recovery of transplanted animals is due, in part, to reinnervation of the striatum. In addition, long-term alterations in receptor density may be related to the behavioral deficits that are associated with the 6-OHDA-lesioned rat. Furthermore, dopamine receptor plasticity may play a role in the functional recovery of substantia nigra transplanted animals and graft viability seems to be a prerequisite for behavioral recovery as well as receptor normalization.