Distribution of adenylate cyclase in the brain of Apteronotus leptorhynchus as revealed by forskolin binding.

An antibody directed against an isoform of the rat regulatory subunit of protein kinase A and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 53 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in fish brains. The 53-kDa band was enriched in fish forebrain. [3H]Forskolin binding was used as a marker for the distribution of adenylate cyclase. [3H]Forskolin binding was nearly completely displaced by excess cold forskolin; specific [3H]forskolin binding sites were heterogenously distributed with relatively high densities in some gray matter regions and low densities in fiber tracts. A high density of [3H]forskolin binding sites was found in the dorsal forebrain with lower densities in most ventral forebrain nuclei. Moderate binding densities were observed in the preoptic and hypothalamic areas with the exception of the nucleus tuberis anterior, which had high levels. The thalamus and midbrain had low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was low. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal and ventral molecular layers, which contain feedback inputs; the cellular layers of the ELL had low binding densities.     

Age-related changes in bindings of second messengers in the rat brain

Age-related alterations in bindings of major second messengers in the brain were studied in 3-week- and 6-, 12-, 18- and 24-month-old Fisher 344 rats using receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin were used to label protein kinase C (PKC) and adenylate cyclase, respectively. In immature rats (3-week-old), [³H]PDBu binding showed a significant decrease only in the cerebellum as compared to adult rats (6-month-old), whereas [³H]forskolin binding exhibited a significant reduction in the neocortex, nucleus accumbens, thalamus and substantia nigra. In aged rats, [³H]PDBu binding showed no significant change in all brain areas. In contrast, [³H]forskolin binding showed a conspicuous reduction in various brain areas in 18-month-old rats as compared to adult animals. The age-related reduction was especially observed in the cerebral cortex, hippocampal CA3 pyramidal cell layer, dentate gyrus, thalamus and molecular layer of cerebellum of 24-month-old rats. The results indicate that adenylate cyclase system in the rat brain is more susceptible to aging processes than phosphoinositide cycle system. Furthermore, our data demonstrate that the change in the adenylate cyclase system is more pronounced than that in the phosphoinositide cycle system in immature rat brain. These findings suggest that the adenylate cyclase system is primarily affected in aging processes and this may lead to age-related neurological deficits.     

Autoradiographic analysis of second-messenger systems in the gerbil brain

Quantitative in-vitro autoradiographic study was performed to localize two prominent second-messenger systems (the adenylate cyclase and phosphoinositide systems) in the normal gerbil brain. [3H] Forskolin and [3H] phorbol 12, 13-dibutyrate (PDBu) were used to identify the regional distribution of adenylate cyclase and protein kinase C, respectively. The localization of the forskolin binding was not uniform, being particularly concentrated in the striatum, the accumbens nucleus, the olfactory tubercle, the substantia nigra, the CA3 region of the hippocampus and the molecular layer of the cerebellum. On the other hand, the PDBu binding was rather uniform, although the superficial layer of the cerebral neocortices, the strata oriens of the CA1 region of the hippocampus and the molecular layer of the cerebellum showed relatively dense binding. Quantitative autoradiography of the second-messenger systems in the brain is expected to provide important information concerning the role of neurotransmitters in the pathophysiology of various conditions.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Localization and Quantitative Autoradiography of Glutamatergic Ligand Binding Sites in Chick Brain

The anatomical localization of glutamate receptor subtype-selective ligand binding sites was investigated in 1-day-old chick brain using quantitative autoradiography. Under the conditions used, the regional distributions of [3H]glutamate, [3H]AMPA (a selective quisqualate receptor ligand) and [3H]kainate binding sites are manifestly different. [3H]l-glutamate binding is densely localized in the telencephalon, particularly in the neostriatum (2.8 pmol/mg protein). In addition, [3H]l-glutamate labels the thalamus, the nucleus mesencephalicus lateralis pars dorsalis, the superficial layers of the optic tectum and the molecular layer of the cerebellum. [3H]AMPA binding sites are most densely localized in the hippocampus (0.90 pmol/mg protein), with an otherwise relatively uniform distribution of binding within the telencephalon. [3H]AMPA also labels the striatum griseum et fibrosum superficiale of the optic tectum and the molecular layer of the cerebellum. [3H]Kainate binding sites are extremely densely packed in the molecular layer of the cerebellum (10 pmol/mg protein). Other regions of [3H]kainate binding include the hyperstriatum and the thalamus. The binding of the NMDA receptor channel blocker [3H]MK-801 is increased in the presence of 1 mM l-glutamate. [3H]MK-801 binding is generally widespread in the telencephalon but is notably absent from the ectostriatum. No evidence of [3H]MK-801 binding sites was detected in the cerebellum, even in the presence of 1 mM l-glutamate. The relatively high densities and the well-defined localizations of the glutamate receptor subtype binding sites suggest that chick brain provides a useful system for the further study of excitatory amino acid receptors.     

Autoradiographic localization of D1 dopamine receptors in the rat brain with [3H]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 (Kd = 2.05 nM); it occurred at a 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.     

Quantitative analysis of the localization of adrenergic binding sites in chick brain

In the present work [3H]-WB4101, [3H]-DHA, and [3H]-clonidine were used for the study of the localization of alpha 1, alpha 2, and beta adrenergic receptors in the chick brain. The highest concentration of [3H]-WB4101 was observed in the nucleus pretectalis, followed by the nucleus brachium conjunctivum descendens. The superficial layers of stratum griseum fibrosum superficiale, the nucleus mesencephalis lateralis pars dorsalis, and the locus coeruleus showed concentrations of [3H]-WB4101 binding higher than 300 fmoles/mg protein. Concentrations of [3H]-DHA binding higher than 300 fmoles/mg protein were observed in the paleostriatum, the external part of nucleus pretectalis, the nucleus isthmi parvocellularis, the nucleus mesencephalis lateralis pars dorsalis, the dorsal nucleus of oculomotor center, and the molecular layer of cerebellum. Locus coeruleus was the only area of chick brain which showed concentration of [3H]-clonidine binding higher than 300 fmoles/mg protein. With few exceptions, [3H]-clonidine binding was very low and in the telencephalon it was undetectable.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Characterization of [3H] CCK4 binding sites in mouse and rat brain

We have investigated the possible occurrence of distinct CCK8 and CCK4 binding sites in the brain by comparing the binding characteristics of [3H] CCK4 to those of the CCK8 analogue, [3H] Boc (Nle28,31]CCK27-33 (BDNL-CCK7). [3H] CCK4 and [3H] BNDL-CCK7 were shown to interact with mouse brain membranes with very similar maximal binding capacities 31.7 +/- 2.1 fmol/mg prot (KD = 3.78 +/- 0.47 nM) and 38.9 +/- 2.2 fmol/mg prot (KD = 0.26 +/- 0.02 nM) respectively. The apparent affinities of five CCK analogues for the sites labelled by both probes were almost identical. Autoradiographic studies revealed that the distribution of [3H] CCK4 binding sites in rat forebrain was the same as that of [3H] BDNL-CCK7, with high densities of receptors in the cortex, nucleus accumbens, olfactory bulb and the medial striatum, moderate densities in the amygdala, the hippocampus, several nuclei of the thalamus and hypothalamus. However in the interpenduncular nucleus where there was moderate binding of [3H]BDNL-CCK7, no [3H]CCK4 labelling was observed. These studies demonstrated the occurrence of one class of high affinity binding sites for [3H] CCK4 in mouse and rat brain, with characteristics similar to those already reported with CCK33, CCK8 and pentagastrin probes. Nevertheless the presence of a small amount of very high affinity binding sites for [3H]CCK4 cannot be excluded.     

High affinity GABA receptors — Autoradiographic localization

The distribution of the high affinity gamma-aminobutyric acid (GABA) receptor labeled by [3H]muscimol, has been studied in the rat brain by light microscopic autoradiography. Receptors in slide-mounted tissue sections were labeled in vitro with [3H]muscimol. Most of the gray matter areas presented grain densities significantly higher than background or white matter areas. Wide variations in receptor densities were found between different brain areas and nuclei. Areas with very high grain densities are the granule cell layer of the cerebellum, external plexiform layer of the olfactory bulb and nuclei of the thalamus, such as the ventral nucleus, lateral nucleus and dorsal geniculate body. The molecular layer of the hippocampus and the external (I-IV) layers of the cortex are also rich in GABA receptors. The basal ganglia have moderate concentrations of receptors, while the pons, medulla and brainstem have only low concentrations of autoradiographic grains. These distributions are discussed in correlation with the known distribution of GABAergic terminals and the presence of inhibitory GABAergic mechanisms.     

Quisqualate- and NMDA-sensitive [3H]glutamate binding in primate brain

Excitatory amino acids (EAA) such as glutamate and aspartate are probably the neurotransmitters of a majority of mammalian neurons. Only a few previous studies have been concerned with the distribution of the subtypes of EAA receptor binding in the primate brain. We examined NMDA- and quisqualate-sensitive [3H]glutamate binding using quantitative autoradiography in monkey brain (Macaca fascicularis). The two types of binding were differentially distributed. NMDA-sensitive binding was most dense in dentate gyrus of hippocampus, stratum pyramidale of hippocampus, and outer layers of cerebral cortex. Quisqualate-sensitive binding was most dense in dentate gyrus of hippocampus, inner and outer layers of cerebral cortex, and molecular layer of cerebellum. In caudate nucleus and putamen, quisqualate- and NMDA-sensitive binding sites were nearly equal in density. However, in globus pallidus, substantia nigra, and subthalamic nucleus, quisqualate-sensitive binding was several-fold greater than NMDA-sensitive binding. In thalamus, [3H]glutamate binding was generally low for both subtypes of binding except for the anterior ventral, lateral dorsal, and pulvinar nuclei. In the brainstem, low levels of binding were found, and strikingly the red nucleus and pons, which are thought to receive glutamatergic projections, had approximately 1/20 the binding observed in cerebral cortex. These results demonstrate that NMDA- and quisqualate-sensitive [3H]glutamate binding are observed in all regions of primate brain, but that in some regions one subtype predominates over the other. In addition, certain areas thought to receive glutamatergic projections have low levels of both types of binding.     

Quantitative light microscopic autoradiographic localization of alpha 2-adrenoceptors in the human brain.

In the present work the anatomical distribution of alpha 2-adrenoceptors in the human central nervous system was studied in detail by quantitative autoradiography using the selective alpha 2 agonist [3H]bromoxidine ([3H]UK-14304) as a ligand. Only postmortem tissues from subjects free of neurological disorders were used in this study. Very high or high densities of alpha 2-adrenoceptors were found along layers I and III in non-visual neocortex, layers III and IVc of the visual cortex, CA1 field--stratum lacunosum-moleculare--and dentate gyrus--stratum granularis--at the hippocampal formation, nucleus arcuatus at the hypothalamus, locus ceruleus, nucleus dorsalis of vagus and at the stratum granularis of the cerebellar cortex. Relevant densities of alpha 2-adrenoceptors were also observed along the remaining layers of neocortex, nuclei centralis, medialis and corticalis at the amygdala, anterior thalamic group and rotundocellularis nuclei, paraventricular and ventromedial hypothalamic nuclei, substantia innominata, superior colliculus--stratum zonale--and lateral periaqueductal area at the midbrain, nucleus tractus solitarii and dorsal horn--substantia gelatinosa--of the spinal cord. [3H]Bromoxidine specific binding was very low or negligible in the remaining brain areas. Although a general parallelism between the distribution of these receptors could be observed for the rat and human brain, dramatic species differences in the level of alpha 2-receptors were found in several brain areas, such as thalamus, amygdala or cerebellar cortex. In general, the distribution of alpha 2-adrenoceptors in the human brain found here was parallel to that described for the noradrenergic presynaptic terminals in the mammalian central nervous system, lending some weight to the proposed predominant presynaptic localization of these receptors. The relevance of the anatomical distribution of alpha 2-adrenoceptors in the human brain for a better knowledge of the neurochemistry of neuropsychiatric disorders is discussed.     

3H]linopirdine (DuP 996) labels a novel binding site in rat brain involved in the enhancement of stimulus-induced neurotransmitter release: autoradiographic localization studies.

A novel high-affinity binding site for linopirdine (DuP 996; 3,3-bis(4-pyrindinylmethyl)-1-phenylindolin-2-one), a cognitive enhancer which improves learning and memory in rodents and primates, has recently been identified in rat brain homogenates. [3H]Linopirdine binding sites were localized in rat brain using in vitro labeling, light microscopic autoradiography. Highest densities of binding sites were present in the hippocampus (CA1 to CA3 pyramidal cell layers and the granule cells of the dentate gyrus), the cerebral cortex (lamina IV), the dorsal raphe nucleus and the interpeduncular nucleus. Moderate densities were present in the olfactory bulb, olfactory tubercle, amygdala, subiculum and medial habenular nucleus. Lower levels of binding were present in the caudate/putamen, thalamus and hypothalamus. The localization of [3H]linopirdine binding sites in brain areas implicated in cognitive processes and affected in Alzheimer's disease suggest that ligands for this binding site may have therapeutic potential for the treatment of cognitive deficits seen in dementia.     

Characterization and autoradiographic distribution of hemicholinium-3 high-affinity choline uptake sites in mammalian brain

[3H]hemicholinium-3 (HC-3) binding characteristics have been investigated using membrane binding assays and in vitro receptor autoradiography. In rat brain membrane preparations, [3H]HC-3 binds with high affinity to an apparent single class of sites. [3H]HC-3 binding is Na+-dependent. The ligand selectivity pattern strongly suggests that [3H]HC-3 selectivity labels the high affinity choline uptake (HACU) in brain membranes (HC-3 greater than choline greater than carbamylcholine greater than acetylcholine). This hypothesis is also supported by quantitative autoradiographic data which demonstrate that the discrete distribution of [3H]HC-3 binding sites correlates very well with the known distribution of other cholinergic markers such as choline acetyltransferase (ChAT), acetylcholinesterase (AChE), HACU, and [3H]AH-5183 (blocker of the vesicular transport of acetylcholine). For example, high densities of labelling are observed for these different markers in the interpeduncular nucleus, anteroventral nucleus of the thalamus, striatum, basolateral nucleus of the amygdala, and an exquisite laminar distribution in the hippocampus. Similar autoradiographic distributions of [3H]HC-3 binding sites are observed in other mammalian species such as guinea pig and monkey. Finally, 7-day unilateral kainic acid lesions of the nucleus basalis magnocellularis (nbm) decrease cortical [3H]HC-3 binding and ChAT activity, although not to a similar extent. In summary, these results demonstrate that [3H]HC-3 is a selective ligand of the HACU in mammalian brain. Thus, it is now possible to characterize precisely various structural components of the cholinergic synapses using markers such as [3H]HC-3, ChAT, HACU, [3H]AH-5183, and selective muscarinic and nicotinic receptor radioligands.     

Heterogeneous localization of protein kinase C in rat brain: autoradiographic analysis of phorbol ester receptor binding

Protein kinase C is a calcium- and phospholipid-stimulated enzyme present in high concentration in the brain. Phorbol esters are potent tumor promoters that bind to specific receptors with high affinity. Several lines of evidence indicate that the phorbol ester receptor is identical to protein kinase C. To determine the distribution of protein kinase C, we have localized phorbol ester receptors in the rat brain by autoradiography, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) and have performed a variety of lesions to assess the nature of the cellular elements possessing the binding sites. The [3H]PDBu binding sites in the rat brain are discretely localized and primarily associated with neurons. Evidence is presented for localization to intrinsic neurons of the cortex and hippocampus, terminals of the striatonigral projection, a projection to the molecular layer of the dentate gyrus, and to dendrites of Purkinje cells.     

Neuroanatomical localization ofκ1and κ2 opioid receptors in rat and guinea pig brain

The neuroanatomical localization of kappa opioid receptors in rat and guinea pig brain was determined by quantitative in vitro receptor autoradiography. Our study shows striking differences in kappa 1 and kappa 2 receptor distributions both between species and within each species. In the rat brain, kappa 1 sites (labeled by [3H]U-69,593) are of low density and confined to a small number of structures. These include the claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, midline nuclear group of the thalamus, superficial grey layer of the superior colliculus, and central grey. kappa 2 sites (labeled by [3H]ethylketocyclazocine or [3H]bremazocine under conditions in which mu, delta, and kappa 1 binding was suppressed) are more widely distributed throughout all levels of rat brain. kappa 2 sites occur at high density in the caudate putamen, nucleus accumbens, amygdala, thalamus, and interpeduncular nuclei. In guinea pig brain, kappa 1 sites predominate and are of high density in layers I and VI of the neocortex, claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, and molecular layer of the cerebellum. As in rat brain, kappa 2 sites in guinea pig are more uniformly and widely distributed throughout the brain than are kappa 1 sites. The highest density of kappa 2 sites is in the dorsal parabrachial nucleus, interpeduncular nuclei, mammillary nuclei, and posterior thalamic nuclei. Results from this study demonstrate important interspecies differences in the distribution of kappa 1 and kappa 2 opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Phorbol ester binding sites in human brain: Characterization,regional distribution,age-correlation,and alterations in Parkinson’s disease

We have characterized and localized phorbol ester binding sites in human autopsied brains, using [³H]phorbol 12,13-dibutyrate ([³H]PDBu). When the tissue was homogenized in the absence of Ca²⁺ chelator (10 mM EGTA/2 mM EDTA), Scatchard analysis of the specific [³H]PDBu bindings to both particulate and soluble fractions yielded a single class of high-affinity binding site (K _d = 7.1 and 7.4 nM:B _max = 45.4 and 3.1 pmol/mg protein, respectively). The particulate fraction retained the majority of [³H]PDBu binding (98% of total binding activity), while the soluble fraction was almost devoid of binding activity (2%). In the presence of Ca²⁺ chelator, more of the activity was found in the soluble fraction (30%). The binding of [³H]PDBu was potently inhibited by active phorbol esters and related diterpenes withK _i of nanomolar concentration but not by inactive ones. Diolein (OAG), a synthetic diacylglycerol, and polymixin B, an inhibitor of protein kinase C (PKC), inhibited the binding moderately (K _i = 5.8 and 1.3 μM, respectively). H-7, an inhibitor of PKC and cyclic nucleotides-dependent kinase, did not compete with [³H]PDBu for the binding sites (K _i > 100,000 nM). The regional distribution of specific [³H]PDBu binding in the human brain was rather uneven and resembled that of [³H]PDBu autoradiograms and PKC-like immunoreactivities in the rat brain. The binding capacities were generally in the order: rhinencephalon > basal ganglia > cerebral cortex > diencephalon > cerebellum > mesencephalon. Age-related loss of binding sites was observed in the prefrontal cortex of the subjects 33–81 years of age. In Parkinson’s disease, the phorbol ester binding showed a significant reduction in the substantia nigra, caudate putamen, and pallidum, whereas it was unchanged in the prefrontal cortex and caudate nucleus of schizophrenics, when compared with the relevant controls. This work was supported by a Grant-in-Aid for Special Project Research of Selected Intractable Neurological Disorders from the Ministry of Education, Science and Culture, Japan.     

Autoradiographic identification and characterization of sigma receptors in guinea pig brain using [3H]1(cyclopropylmethyl)-4-(2'-(4'-fluorophenyl)-2'-oxoethyl) piperidine ([3H]DuP 734), a novel sigma receptor ligand.

The psychotomimetic effects of certain cycloalkyls and benzomorphans that interact with sigma receptors has led to the hypothesis that these sites may be important in the etiology of schizophrenia. DuP 734 [1-(cyclopropylmethyl)-4-(2'-(4'-fluoro-phenyl)-2'-oxoethyl) piperidine HBr] is a novel sigma receptor ligand. The receptor binding specificity and neuroanatomical distribution of [3H]DuP 734-labeled sigma receptors in guinea pig brain were examined using quantitative autoradiography. [3H]DuP 734 binding (10 microM haloperidol displaceable) to slide-mounted sections of guinea pig brain was saturable and of high affinity (Ki = 3.9 nM). Competition studies, under conditions identical to those used to visualize the receptor, yielded the following rank order of potency: DuP 734 > haloperidol > (+)-pentazocine > (-)-butaclamol > DTG > (+)-SKF 10,047 > (+)-3-PPP > (-)-pentazocine > (+)-butaclamol > U50,488H > (-)-SKF 10,047 > cinanserin > PCP > MK801, sulpiride. High densities of [3H]DuP 734 binding sites displaceable by haloperidol were present in the limbic system, in particular the dorsal and ventral bands of Broca as well as the ventral pallidum. Within the hippocampus, the pyramidal layers were sparsely labeled, while higher densities of binding sites were evident in the dentate gyrus. The frontal cortex, the mammillary complex of the hypothalamus, the central gray and red nucleus of the midbrain, the pontine reticular nucleus, the Purkinje cell layer of the cerebellum and dorsal and ventral horns, as well as the central gray matter of the spinal cord, all showed enrichments of [3H]DuP 734 binding sites. Lower levels of binding were present in the other regions of the cerebral cortex including parietal, pyriform, occipital, cingulate cortex, as well as the basal ganglia, and negligible specific binding was present in the white matter tracts. The kinetic and pharmacological characteristics and distribution of [3H]DuP 734 binding sites in brain are similar to those previously reported for sigma receptors.     

3H]vesamicol binding in brain: autoradiographic distribution, pharmacology, and effects of cholinergic lesions

An autoradiographic analysis of high-affinity binding sites for the vesicular acetylcholine transport blocker [3H]vesamicol (2-(4-phenylpiperidino) cyclohexanol; AH 5183) was conducted in rat brain. [3H]Vesamicol binding was displaced 52-99% by DPPN [( 2,3,4,8]-decahydro-3-(4-phenyl-1-piperidinyl)-2-napthalenol) (IC50 = 14 nM) and by ketanserin (500 nM), haloperidol (43 nM), and vesamicol analogs, but not by drugs selective for adenosine, adrenergic, amino acid, calcium channel, monoaminergic, opioid, PCP, sigma, or several other receptor classes. [3H]Vesamicol binding was most concentrated in the interpeduncular nucleus and fifth and seventh cranial nerve nuclei. Moderate binding was found in the lateral caudate-putamen, medial nucleus accumbens, olfactory tubercle, vertical and horizontal diagonal bands of Broca, and basolateral amygdala. The distribution of [3H]vesamicol binding was similar to distributions of acetylcholine (r = 0.88), acetylcholine esterase (r = 0.97), choline acetyltransferase (ChAT) (r = 0.97), and [3H]hemicholinium-3 binding sites (r = 0.95-0.99). Lower correlations were obtained between [3H]vesamicol and muscarinic receptor densities (r = 0.50-0.70). Few exceptions to the match between binding and cholinergic neuronal markers were found, e.g., the molecular layer of the cerebellum and the thalamus. Lesions of cholinergic neuronal projections to the neocortex or hippocampus reduced [3H]vesamicol binding in each of these regions, but to a lesser extent than reductions in ChAT. [3H]Vesamicol binding sites appear to be anatomically associated with brain cholinergic neurons, a locus that is consistent with the control by this site of vesicular acetylcholine uptake.