Quantitative light microscopic autoradiographic localization of α2-adrenoceptors in the human brain

In the present work the anatomical distribution of α₂-adrenoceptors in the human central nervous system was studied in detail by quantitative autoradiography using the selective α₂ 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 α₂-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 α₂-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 α₂-receptors were found in several brain areas, such as thalamus, amygdala or cerebellar cortex. In general, the distribution of α₂-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 α₂-adrenoceptors in the human brain for a better knowledge of the neurochemistry of neuropsychiatric disorders is discussed.     

Quantitative light microscopic localization of [H]naltrindole binding sites in the rat brain

The binding of radiolabeled naltrindole ([³H]NTI), a selective δ-opioid antagonist, was characterized using receptor autoradiography. Receptor binding properties were established in brain paste slices which demonstrated one site receptor occupancy with an apparent K_d of 0.25 ± 0.08nM (B_max of 597.5 fmol/mg protein). Autoradiographic localization of [³H]NTI binding sites in the rat brain revealed high densities of these sites in the cortex (layers 1–3 and 6), caudate putamen, accumbens, claustrum, and internal plexiform layer of the olfactory bulb. Moderate to low levels of specific binding were observed in the hippocampus, thalamus, and substantia gelatinosa of the spinal cord.     

Coupling of human brain cerebral cortical alpha 2-adrenoceptors to GTP-binding proteins in Alzheimer's disease.

The coupling of alpha 2-adrenoceptors to guanine nucleotide binding G-proteins was investigated in cerebral cortical membranes from control and Alzheimer's disease brain by characterizing the effects of MnCl2 and Gpp[NH]p on [3H]clonidine binding. The manganese induced elevation of [3H]clonidine binding was apparent in both control and Alzheimer's disease samples and the effect showed no significant difference between the two groups in the frontal cortex. However, the MnCl2 concentration dependence curves for control and Alzheimer's disease samples were significantly different from one another in the temporal cortex, whereas the pattern of stimulation by MnCl2 remained the same. The guanine nucleotide analogue, Gpp[NH]p inhibited [3H]clonidine binding in a concentration-dependent manner, the profiles of inhibition showing no significant differences between control and Alzheimer's disease samples. Analysis of the effect of Gpp[NH]p on [3H]clonidine saturation binding curves showed no significant differences between control and Alzheimer's disease samples in either frontal (Kd = 9.68 +/- 1.38, 9.1 +/- 2.6 nM; Bmax = 40.23 +/- 4.33, 44.3 +/- 9.4 fmol/mg, control and Alzheimer's disease values, respectively), or temporal (Kd = 11.61 +/- 4.04, 5.38 +/- 2.5 nM; Bmax = 52.0 +/- 14.0, 31.07 +/- 8.00 fmol/mg control and Alzheimer's disease values, respectively) cortices.(ABSTRACT TRUNCATED AT 250 WORDS)     

β-Adrenoceptor subtypes in the human brain: autoradiographic localization

The distribution and characteristics of beta-adrenoceptors in postmortem human brain was studied using quantitative autoradiographic techniques. 125I-Cyanopindolol was used as a ligand. High densities of beta-adrenoceptors were found in the caudate, putamen, different cortical areas and layers and the hippocampal formation. Low densities were present in other areas such as the thalamus, hypothalamus, midbrain and cerebellar cortex. Specific beta 1 and beta 2 antagonists were used to visualize and quantify separately the two subtypes of beta-adrenoceptors. Computer analysis of the competition curves obtained revealed that the putamen was enriched in beta 1 sites while the cerebellum contained predominantly beta 2 adrenoceptors. The regional distribution of beta-adrenoceptor subtypes was found to be similar to that seen in the rat brain.     

Quantitative autoradiographic localization of the D1 and D2 subtypes of dopamine receptors in rat brain

The distribution of D1 and D2 receptors was studied in coronal sections of rat brain, using quantitative autoradiography. D1 receptors were labeled with 1.8 nM 3H-SKF-83566 (a brominated analog of 3H-SCH-23390), while D2 receptors were labeled with 1.0 nM 3H-spiroperidol (3H-SPD). The binding of both ligands to sections from brain and from a homogenate of caudate putamen (CPu mash) reached equilibrium within 80 min at 37 degrees C. CPu mash provided a virtually unlimited number of homogeneous sections that contained a high density of both D1 and D2 receptors. Sections of CPu mash were used in competition studies that confirmed that the specific binding of 3H-SKF-83566 was selective for D1 receptors, and that the binding of 3H-SPD was selective for D2 receptors. Scatchard analysis of equilibrium binding of the 2 ligands in the CPu in horizontal sections of rat brain revealed Kd values of 1.1 +/- 0.07 nM for 3H-SKF-83566 and 0.7 +/- 0.09 nM for 3H-SPD. Studies of the distribution of D1 and D2 receptors were carried out in coronal sections of brains from 5 rats. D1 receptors were found throughout the forebrain and were present in greater density than were D2 receptors in all regions examined except the olfactory nerve layer. In the CPu, nucleus accumbens, and olfactory tubercle, the densities of D1 and D2 receptors were, respectively, approximately 2,500 and 600-800 fmol/mg protein. In the substantia nigra, the density of D1 receptors was approximately 2,500 fmol/mg protein in both the compacta and the reticulata, but the density of D2 receptors was 230 fmol/mg protein in the compacta and 70 fmol/mg protein in the reticulata. The ventral tegmental area contained only 90 fmol/mg protein of D1 receptors, and D2 receptors were undetectable. The entopeduncular nucleus, zona incerta, and region of the ventral internal capsule had densities of D1 receptors of 550-950 fmol/mg protein and D2 receptor densities of less than 100 fmol/mg protein. Densities of D1 and D2 receptors were, respectively, 2,700 and 900 fmol/mg protein in the choroid plexus. Knowledge of the differences in the relative distributions of D1 and D2 receptors in various brain regions may increase our understanding of the functions of brain dopaminergic systems and may aid in the development of new therapeutic approaches for neuropsychiatric disorders.     

Glycine receptor: light microscopic autoradiographic localization with [3H]strychnine

Glycine receptors have been localized by autoradiography in the rat central nervous system (CNS) using [3H]strychnine. The gross distribution of receptors is in excellent accord with the distribution determined by filtration binding assays. Specifically, the density of glycine receptors is greatest in the gray matter of the spinal cord and decreases progressively in regions more rostral in the neuraxis. Glycine receptors were found to be associated with both sensory and motor systems in the CNS. Moreover, there is a striking correlation between areas of high strychnine binding site density and areas in which glycine has been found to be electrophysiologically active. Finally, the anatomic localization of strychnine binding sites may help explain many of the signs and symptoms of strychnine ingestion. For example, individuals consuming subconvulsive doses of strychnine frequently experience altered cutaneous and auditory sensation. We have localized strychnine receptors in areas of the acoustic system known to influence discriminative aspects of audition and in areas of the spinal cord and trigeminal nuclei which modulate discriminative aspects of cutaneous sensation. The alteration of visceral functions (e.g., blood pressure and respiratory rate) associated with strychnine ingestion may be accounted for in a similar manner.     

Neurotensin receptor localization by light microscopic autoradiography in rat brain

Neurotensin is a putative peptide neurotransmitter in the mammalian brain. The light microscopic localization of the neurotensin receptor has been carried out by the autoradiographic localization of [3H]neurotensin binding sites in slide-mounted tissue sections. Large variations in receptor distribution were found, even in small areas. In many regions, the distribution of the receptor paralleled that of the immunoreactive neurotensin found in other studies. The results suggest sites of neurotensin's action in producing various physiological effects such as hypothermia and analgesia.     

Quantitative autoradiographic localization of [125I]alpha-bungarotoxin binding sites in the honeybee brain

Binding sites for the nicotinic acetylcholine receptor antagonist, [125I]alpha-bungarotoxin, were localized in the honeybee brain by in vitro autoradiography. Highest binding site densities were localized in the suboesophageal ganglion, the optic tubercles, optic lobes medulla and lobula, antennal lobes, dorsal lobes and the alpha-lobes of the mushroom bodies. The distribution pattern of these putative nicotinic acetylcholine receptors suggests that acetylcholine is involved in several sensory pathways and in central information processing in the honeybee brain.     

Neuronal and glial localization of alpha(2A)-adrenoceptors in the adult zebrafish (Danio rerio) brain

The alpha(2A)-adrenoceptor (AR) subtype, a G protein-coupled receptor located both pre- and postsynaptically, mediates adrenaline/noradrenaline functions. The present study aimed to determine the alpha(2A)-AR distribution in the adult zebrafish (Danio rerio) brain by means of immunocytochemistry. Detailed mapping showed labeling of alpha(2A)-ARs, in neuropil, neuronal somata and fibers, glial processes, and blood vessels. A high density of alpha(2A)-AR immunoreactivity was found in the ventral telencephalic area, preoptic, pretectal, hypothalamic areas, torus semicircularis, oculomotor nucleus (NIII), locus coreruleus (LC), medial raphe, medial octavolateralis nucleus (MON), magnocellular octaval nucleus (MaON), reticular formation (SRF, IMRF, IRF), rhombencephalic nerves and roots (DV, V, VII, VIII, X), and cerebellar Purkinje cell layer. Moderate levels of alpha(2A)-ARs were observed in the medial and central zone nuclei of dorsal telencephalic area, in the periventricular gray zone of optic tectum, in the dorsomedial part of optic tectum layers, and in the molecular and granular layers of all cerebellum subdivisions. Glial processes were found to express alpha(2A)-ARs in rhombencephalon, intermingled with neuronal fibers. Medium-sized neurons were labeled in telencephalic, diencephalic, and mesencephlic areas, whereas densely labeled large neurons were found in rhombencephalon, locus coeruleus, reticular formation, oculomotor area, medial octavolateralis and magnocellular octaval nuclei, and Purkinje cell somata. The functional role of alpha(2A)-ARs on neurons and glial processes is not known at present; however, their strong relation to the ventricular system, somatosensory nuclei, and nerves supports a possible regulatory role of alpha(2A)-ARs in autonomic functions, nerve output, and sensory integration in adult zebrafish brain.     

Quantitative autoradiographic localization of prostaglandin E2 binding sites in monkey diencephalon

Quantitative autoradiography was performed to investigate the mapping of prostaglandin E2 binding sites in the Macaca fuscata fuscata diencephalon. Autoradiographs were prepared by incubation of 10-micron-thick serial frozen sections with 3H-prostaglandin E2 and were processed by using a rotating drum-scanner and a computer-assisted image-processing system with 3H-microscales as standards. The localization of prostaglandin E2 binding sites was remarkably discrete in the diencephalon. The highest concentrations were found in the median and medial preoptic areas, supramammilary nucleus of the hypothalamus, and centromedian nucleus of the thalamus. High density was observed in the medial and dorsal hypothalamic areas; paraventricular, anterior, dorsomedial, and infundibular nuclei of the hypothalamus; and in the anteroventral, periventricular, paraventricular, laterodorsal, and habenular nuclei of the thalamus. The distribution correlates well with the known effects of prostaglandin E2 and may also give us useful clues in unveiling the novel role of prostaglandin E2 in a variety of brain functions.     

Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors

The distribution of serotonin-2 (5-HT2) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with four ligands: [3H]ketanserin, [3H]mesulergine, [3H]LSD and [3H]spiperone, which are reported to show high affinity for 5-HT2 receptors. Co-incubation with increasing concentrations of several well-known 5-HT2-selective drugs, such as pirenperone, cinanserin and ketanserin, resulted in an inhibition of the binding of the four 3H-labeled ligands to the same areas. However, all of them recognized, in addition to 5-HT2 sites, other populations of binding sites. Receptor densities were quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Our results reveal a heterogeneous distribution of 5-HT2 receptor densities in the rat brain. Very high concentrations were localized in the claustrum, olfactory tubercle and layer IV of the neocortex. The anterior olfactory nucleus, piriform cortex and layer I of neocortex were also rich in 5-HT2 receptors. Intermediate concentrations of receptors were found in caudate putamen, nucleus accumbens, layer V of neocortex, ventral dentate gyrus and mammillary bodies. Areas containing only low concentrations of receptors included the thalamus, hippocampus, brainstem, medulla, cerebellum and spinal cord. The specificity of the different ligands used is discussed in terms of the other populations of sites recognized by them. The distribution of 5-HT2 receptors here reported is discussed in correlation with (a) the known distribution of serotoninergic terminals, (b) the specific anatomical systems and (c) the central effects reported to be mediated by 5-HT2-selective drugs.     

Acute cold-restraint stress affects alpha 2-adrenoceptors in specific brain regions of the rat

The effect of acute cold-restraint stress on binding of [3H]rauwolscine to alpha 2-adrenoceptors was investigated in 10 regions of rat brain. Acute stress: significantly decreased the density but had no significant effect on the affinity of binding sites in the hippocampus; decreased density and increased affinity in the amygdala; and increased density and decreased the affinity in the midbrain. Seven other brain regions showed no significant changes in binding parameters in response to stress. These results, together with previous findings in this laboratory showed that alteration by restraint stress of noradrenaline levels in amygdala and hippocampus, but not other regions, indicate an association between neurotransmitter turnover and receptor function.     

Quantitative autoradiographic distribution of gamma-hydroxybutyric acid binding sites in human and monkey brain

gamma-Hydroxybutyric acid (GHB), a naturally occurring metabolite of GABA, is present in micromolar concentrations in various areas of the mammalian brain. Specific GHB binding sites, uptake system, synthetic and metabolizing enzymes have been identified in CNS. The present study shows the anatomical distribution of GHB binding sites in sections of primate (squirrel monkey) and human brain by radioligand quantitative autoradiography. In both species the highest densities of binding sites were found in the hippocampus, high to moderate densities in cortical areas (frontal, temporal, insular, cingulate and entorhinal) and low densities in the striatum; no binding sites were detected in the cerebellum. High density of GHB binding was found in the monkey amygdala. In addition the binding characteristics of [(3)H]GHB to membrane preparations of human brain cortex were examined. Scatchard analysis and saturation curves revealed both a high (K(d1) 92+/-4.4 nM; B(max1) 1027+/-110 fmol/mg protein) and a low-affinity binding site (K(d2) 916+/-42 nM; B(max2) 8770+/-159 fmol/mg protein). The present study is the first report on the autoradiographic distribution of specific GHB binding sites in the primate and human brain: such distribution is in both species in good agreement with the distribution found in the rat brain.     

Effects of cortisol on brain alpha2-adrenoceptors: potential role in stress

It has been proposed that behavioural changes induced by chronic psychosocial stress in male tree shrews might be related to alterations in the central nervous alpha2-adrenoceptor system. In the noradrenergic centres of the brain, alpha2-adrenoceptors function as autoreceptors regulating noradrenaline release. Chronic stress downregulates these receptors in several brain regions. Since during stress, the activity of the hypothalamus-pituitary-adrenal axis is increased leading to high concentrations of plasma glucocorticoids, we investigated whether the effects of chronic stress can be mimicked by cortisol treatments. Two experiments were performed: a short-term treatment (males were injected i.v. with 1.5 mg cortisol and brains were dissected 2 h later) and a long-term treatment (animals received the hormone in their drinking water for 5 days; daily uptake 3-7 mg). The short-term treatment (injection), similar to the stress effects, downregulated alpha2-adrenoceptors in several brain regions. In contrast, the long-term oral treatment induced regional receptor upregulation. These data show: (i) that glucocorticoids regulate alpha2-adrenoceptors in the brain; (ii) that the duration and/or the route of cortisol application determines the results: and (iii) that chronic stress effects are not only due to the long-term glucocorticoid exposure, but also to other elements of the stress response.     

Autoradiographic characterization of alpha(2C)-adrenoceptors in the human striatum

Indirect experimental evidence suggests that drugs acting on the alpha(2C)-adrenoceptor could be useful in the treatment of neuropsychiatric disorders such as depression and schizophrenia. In rodent brain, the highest levels of alpha(2C)-adrenoceptors are found in the striatum, with lower levels in cerebral cortex and hippocampus. In human brain, because of the poor subtype-selectivity of the available alpha(2)-adrenoceptor ligands, the localization of alpha(2C)-adrenoceptors has remained unknown. Recently, a selective alpha(2C)-adrenoceptor antagonist, JP-1302, was characterized, and to assess the presence of alpha(2C)-adrenoceptors in human brain, we performed competition binding in vitro receptor autoradiography with JP-1302 and the alpha(2)-adrenoceptor subtype nonselective antagonist [ethyl-(3)H]RS79948-197 on rat and human postmortem brain sections. In striatum of both species, JP-1302 vs. [ethyl-(3)H]RS79948-197 competition binding was biphasic, identifying high- and low-affinity binding sites, whereas in cortex and cerebellum, only low-affinity binding sites were detected. The results indicate that a significant portion of the alpha(2)-adrenoceptors in striatum is of the alpha(2C) subtype, whereas non-alpha(2C)-adreocneptors predominate in cortex and cerebellum. Because the alpha(2C)-adrenoceptor subtype distribution pattern appears to be conserved between rodents and humans, results obtained from studies on the role of the alpha(2C)-adrenoceptor in rodent models of neuropsychiatric disorders may be relevant also for human diseases.     

Specific decrease of high-affinity agonist states of alpha2-adrenoceptors in the aging mouse brain

Summary Using the specific binding of the full alpha₂-adrenergic agonist³H-UK-14,304 the contribution of high-affinity agonist states to the total number of alpha₂-adrenoceptors as labeled by the specific binding of the antagonist³H-yohimbine has been investigated in the brain of young and aged mice. In contrast to findings with human platelet membranes, in young mice all central alpha₂-adrenoceptors are present in a high-affinity agonist conformation. While the total number of alpha₂-adrenoceptors was not changed in the brain of aged animals, a specific decline of the high-affinity agonist sites by about 30% was observed. It is suggested that the specific decrease of high-affinity agonist sites of central alpha₂-adrenoceptors might represent one of the mechanisms leading to a general impairment of central noradrenergic neurotransmission with aging.     

Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of 5-HT1C and 5-HT2 recognition sites

The presence, pharmacological properties and anatomical distribution of serotonin-1C and serotonin-2 receptor subtypes were studied in the human brain by both radioligand binding and autoradiographic procedures. Frontal cortex, hippocampus and choroid plexus from human brains obtained at autopsy without history of neurological diseases were used in this study. [3H]5-HT and [3H]mesulergine were used to label 5-HT1C recognition sites while [3H]ketanserin was used to label 5-HT2 receptors. The pharmacological profile of 5-HT1C sites which are very concentrated in the choroid plexus, was extremely similar to that of pig and rat 5-HT1C sites. These receptors were also detected in the hippocampus and the cortex from human brain. The general distribution of 5-HT1C sites in human and rat brain was similar although slight differences were observed. Human 5-HT2 receptors were concentrated in cortical areas but also found in the hippocampus. The pharmacological profile of these receptors was extremely similar in human and pig brain tissue, but differed in certain respects to that found in rat brain 5-HT2 receptors. The anatomical distribution of 5-HT2 receptors is similar in human and rat brain with some differences at the microscopic level. The importance of species differences in the development of 5-HT2 compounds is discussed.     

Muscarinic cholinergic receptor subtypes in the rat brain. I. Quantitative autoradiographic studies

The binding characteristics of N[³H]methylscopolamine (³H]NMS) to slide-mounted tissue sections were studies using quantitative autoradiography. Binding of [³H]NMS was saturable, reversible of high affinity (K_d = 0.26nM). The inhibition of [³H]NMS binding produced by several muscarinic agonists and antagonists was analyzed in 29 discrete brain regions by constructing complete displacement curves. Comparison of IC₅₀ values obtained both biochemically and by autoradiography demonstrated to a very close agreement, supporting the validity of the autoradiographic approach. The competition curves for the agonists carbachol, oxotremorine and 2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion-hydrobromide (RS 86) fitted to a two-site model, with comparable affinity values from region to regions, although different proportions of high- and low-affinity sites were seen in the different areas studied. The distribution of high- and low-affinity sites was similar for the three agonists. Atropine showed monophasic curves presenting similar affinities in all regions studied. In contrast, pirenzepine differentiated between high- and low-affinity sites which showed a distribution opposite to that observed for the agonists. Gallamine, a ligand for a putative regulatory site in the muscarinic receptor, inhibited [³H]NMS binding in a biphasic manner. The calculated IC₅₀ values for the gallamine high- and low-affinity sites did not vary from region to region and the distribution of these sites correlated well with that observed for the agonists. High-affinity pirenzepine sites (also called M₁ sites) were localized mainly in forebrain areas, such as striatum, hippocampus and cortex, and their regional distribution correlated with that of the low-affinity sites for the agonists and gallamine. On the other hand, low-affinity sites for pirenzepine (named M₂ sites) were mainly found in the brainstem and parts of the thalamus. A good correlation was found between pirenzepine low-affinity sites and agonist and gallamine high-affinity sites. The significance of these findings is discussed in relation to the known and possible effects of selective M₁ and M₂ centrally acting agents.