Li-Qun Fan

μ-, δ- and κ-opioid receptor populations are differentially altered in distinct areas of postmortem brains of Alzheimer’s disease patients

Abstract The putative role of the opioid system in cognitive and memory functions prompted us to search for possible changes in the cohort of the major opioid receptors, μ, δ and κ, in Alzheimer’s disease. The present study examines alterations in opioid receptor levels by quantitative autoradiography. These experiments were carried out on coronal sections of postmortem brains from Alzheimer’s disease patients and from aged-matched, dementia-free individuals. Brain sections were labeled with the tritiated forms of μ-, δ- and κ-opioid ligands; DAMGO ([ d -Ala2,N-Me-Phe4,Gly-ol5]-enkephalin), DPDPE ([ d -Pen2,5]-enkephalin) and bremazocine (in the presence of μ- and δ-ligands), respectively. Nonspecific binding was determined in the presence of naloxone (10 μM). Brain areas analyzed were caudate, putamen, amygdaloid complex, hippocampal formation and various cerebral and cerebellar cortices. Image analyses of autoradiographs show, that in comparison to the same areas in control brain, statistically significant reductions in μ-opioid receptor binding occur in the subiculum and hippocampus of Alzheimer’s disease brains. Binding of δ-opioid receptors is also decreased in the amygdaloid complex and ventral putamen of Alzheimer’s disease brains. In contrast, large increases of κ-opioid receptor binding are found in the dorsal and ventral putamen as well as in the cerebellar cortex of Alzheimer’s disease brains. Levels of μ-, δ- and κ-opioid receptor binding are unaltered in the caudate, parahippocampal gyrus and occipito–temporal gyrus. These results may suggest an involvement of the endogenous opioid system in some of the multitude of effects that accompany this dementia.

Laminar distribution of the multiple opioid receptors in the human cerebral cortex

Quantitative autoradiographic assessment of cerebral cortical laminar distribution of μ, δ and κ opioid receptors was carried out in coronal sections of five post-mortem human brains obtained at autopsy. The cortical areas studied were: cingulate, frontal, insular, parietal, parahippocampal, temporal, occipitotemporal, occipital and striate area. In general, the laminar patterns of distribution for the three types of receptors are distinctive. Peak levels of δ opioid binding are in laminae I, II, and IIa. μ-Receptors are located in lamina III followed by I and II in cingulate, frontal, insular and parietal cortices and lamina IV in temporal and occiptotemporal cortices. κ-Receptors are found concentrated in laminae V and VI. The patterns of opioid binding in cortical laminae showed remarkable consistency in all five brains examined. In contrast to other cortical areas, the parahippocampal gyrus, at the level of the amygdaloid formation, demonstrated peak κ receptor density in laminae I, II and III. μ-Opioid binding was undetectable in the lateral occipital cortex and in the striate area.

Lesioning of the nucleus basalis of Meynert has differential effects on mu, delta and kappa opioid receptor binding in rat brain: a quantitative autoradiographic study

Opioid receptor binding was investigated in rat brain following lesioning of the nucleus basalis of Meynert (nbM). The nbM, which provides cholinergic input to the cortex, was lesioned unilaterally using ibotenic acid. The efficacy of lesioning was confirmed by the observation of a significant decrease in choline acetyltransferase (ChAT) activity in the ipsilateral prefrontal cortex. The specific laminar and regional distribution of mu, delta and kappa opioid receptor binding was quantitated in various cortical and limbic structures in the rat using autoradiography. Distinct medial to lateral gradients of mu and kappa opioid binding were observed in regions of the cerebral cortex. In the lesioned hemisphere the levels of mu, delta and kappa opioid binding were altered in localized areas of the cerebral cortex and the hippocampus. The direction of these binding changes varied with the opioid receptor type being assessed. Delta opioid binding was increased in the lateral portions of the frontal, occipital, perirhinal and retrosplenial granular cortices. Kappa opioid binding was increased in the lateral portion of the occipital cortex and in the CA3 region of the hippocampus. In contrast, mu opioid binding was decreased in the lateral portions of the frontal, entorhinal and forelimb cortices. These opioid receptor changes are discussed with respect to the interactions between the cholinergic and opioid systems, and relevance of the nbM lesion model to Alzheimers disease.

Autoradiographic comparison of [3H]DPDPE and [3H]DSLET binding : evidence for distinct δ1 and δ2 opioid receptor populations in rat brain

The delta opioid ligands, [3H]DPDPE (S1) and [3H]DSLET (δ2) were used in quantitative autoradiographic experiments to ascertain whether separate populations of delta opioid subtypes could be identified in rat brain. Densitometric image analysis showed a general similarity in δ1 and δ2 distributions. However, statistically significant differences in binding levels were observed in anatomically discrete regions. Examples of these regions and their δ2/δ1 ratio(s) are: dorsomedial hypothalamus (9.3), ventromedial hypothalamus (4.9), superior colliculis (2.7), medial division of bed nucleus stria terminalis (1.6–3.0), external cortex of the inferior colliculis (2.1), amygdaloid nuclei (1.5–2.1), cingulate cortex (1.8), CAI, CA2, and CA3 regions of Ammons horn (1.6–2.0), dentate gyrus (1.7), laminar VI of the frontal, forelimb, hindlimb and parietal cortices (1.3–1.8), nucleus accumbens (1.4) and caudate/putamen (1.3). These findings provide evidence supporting the existence of distinct δ1 and δ2 opioid receptors.

Functional Reconstitution of a Highly Purified μ-Opioid Receptor Protein with Purified G Proteins in Liposomes

Abstract: A μ‐opioid receptor protein (μ‐ORP) purified to homogeneity from bovine striatal membranes has been functionally reconstituted in liposomes with highly purified heterotrimeric guanine nucleotide regulatory proteins (G proteins). A mixture of bovine brain G proteins, predominantly GoA, was used for most of the experiments, but some experiments were performed with individual pure G proteins, GoA, GoB, Gi1, and Gi2. Low Km GTPase was stimulated up to 150% by μ‐opioid receptor agonists when both μ‐ORP and a G protein (either the brain G protein mixture or a single heterotrimeric G protein) were present in the liposomes. Stimulation by a selective μ‐agonist was concentration dependent and was reversed by the antagonist (−)‐naloxone, but not by its inactive enantiomer, (+)‐naloxone. The μ selectivity of μ‐ORP was demonstrated by the inability of δ and κ agonists to stimulate GTPase in this system. High‐affinity μ‐agonist binding was also restored by reconstitution with the brain G protein mixture and with each of the four pure Gi and Go proteins studied. The binding of μ agonists is sensitive to inhibition by GTPγS and by sodium.

Age-related changes in kappa opioid receptors in the guinea-pig brain: A quantitative autoradiographic study

Investigation into the effect of aging on kappa opioid receptors in the brains of guinea-pigs was carried out in animals aged one, six, 24 and 36 months. Quantitative autoradiography was used to monitor the concentration of kappa receptors in various anatomic regions at five rostrocaudal levels in each age-group. Areas of high binding were found in the deep layers (laminae V, VI) of the neocortex and in the internal band of the periallocortical, dorsal agranular insular cortex. Among non-cortical areas examined, the nucleus accumbens and the substantia nigra possessed kappa binding levels equal to those seen in the deep neocortical layers. In all cases where an age-related change in the level of kappa receptors was detected, the direction of the change was one of decreased binding with advancing age. Statistical analysis of the binding data revealed that the one-month-old animal possessed the highest levels of kappa binding among all age groups examined. The vast majority of age-related changes in kappa binding levels occurred in laminae V and VI of neocortical regions. The per cent decreases (18-42%), as well as their age of onset (six to 36 months) varied in different anatomical regions. Possible mechanisms to explain the age-related decreases in kappa opioid binding are presented. The majority of the age-related decreases in kappa opioid binding are found in areas of the neocortex which are characterized by their motor, sensory and associative functions. It is within these three areas of function that diminutions in performance are most apparent in senescence.

Photoaffinity ligands for the MU opioid receptor

Two affinity ligands, 6 beta-(5-Azido-2-nitrophenacetamido) 14 beta-hydroxy-7,8-dihydromorphinone (4) and 6 beta-(5-azido-2-nitrophenacetamido) 14 beta-hydroxy-7,8-dihydro-N- cyclopropylmethylnormorphinone (5) bind reversibly to opioid receptors present in bovine caudate membranes and photolyse in a range of wavelengths centered about 366 nm to produce wash-resistant binding to the mu receptor. At these wavelengths very little if any photodestruction of the mu receptor occurs over the 20 minute period of irradiation at 0 degree C.

Alterations in δ opioid receptor levels in discrete areas of the neocortex and in the globus pallidus of the aging guinea pig: a quantitative autoradiographic study

Abstract The effect of aging on δ opioid receptors was examined in the brains of guinea pigs aged 1, 6, 24 and 36 months. Quantitative autoradiography was used to monitor the concentrations of δ receptors in various anatomical regions at five rostro-caudal levels. δ opioid receptor populations were found to be remarkably stable throughout the life span of this species. We have, however, discovered anatomical areas which offer striking exceptions. In the globus pallidus, progressive age-related losses of δ receptors reached 50% in the senescent animal. In contrast, laminae I, II of the lateral agranular frontal cortex and laminae I, II and III, IV of the primary somatosensory cortex demonstrated age-related increases in the concentrations of δ receptors ranging from 30 to 45%. These changes are discussed with the view to their being functionally related components of motor circuitry involving pyramidal and extrapyramidal elements.

Differential effects of cyanogen bromide on ligand binding by mu, delta and kappa opioid receptors

Guinea pig brain membranes treated with cyanogen bromide (CNBr) demonstrate a loss in the number of mu opioid receptors and a lower binding affinity of delta opioid receptors. These receptor changes are irreversible. Results from ligand protection experiments support the hypothesis that the location of the methionine groups, the sites at which CNBr cleaves peptides, differs between these two types of opioid receptors. Kappa receptors are significantly less sensitive to the action of CNBr than mu or delta receptors.

Reconstitution in liposomes of a μ-opioid binding protein purified to homogeneity from bovine striatal membranes

Abstract An opioid binding protein (OBP) purified to homogeneity from bovine striatal membranes was reconstituted into liposomes. For most experiments a CHAPS extract of bovine striatum, devoid of opioid binding, served as source of G-proteins and lipids. Liposomes were formed by precipitation of a mixture of OBP and CHAPS extract with polyethylene glycol-6000 (PEG). Reconstituted OBP bound μ agonist ligands stereos-pecifically and with high affinity, similar to that of membrane-bound μ-receptors. The binding was highly selective for μ-ligands, as compared to δ and κ-ligands and was completely inhibited by GTPγS. Similar results were obtained in reconstitution experiments with purified G-proteins. Stimulation of low Km GTPase by μ-agonists was observed. These results indicate that recoupling of purified receptor with G protein has taken place in this system and confirm that OBP is a μ binding protein.