Jacob M. Hiller

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.

Selective changes in μ, δ and ϰ opioid receptor binding in certain limbic regions of the brain in Alzheimer's disease patients

Abstract Total opioid binding and levels of the three major types of opioid binding sites were measured in homogenates of various limbic structures from post-mortem brains of Alzheimers disease patients and age-matched control individuals. The most consistent finding in Alzheimers disease brains was an increase in ϰ binding in all 6 areas of the limbic system examined, with the putamen and caudate regions showing significant increases of 114% and 53%, respectively. In addition, the Alzheimers disease putamen showed a significantly higher level of total binding (85% increase). The amygdala of Alzheimers disease patients exhibited significantly lower levels of μ and δ binding (41% and 55% decrease, respectively). Total binding and binding to μ and δ receptors in frontal cortex, caudate and hippocampus of Alzheimers disease brains was indistinguishable from levels seen in these brain areas from control individuals.

Lectin binding of solubilized opiate receptors: Evidence for their glycoprotein nature

Abstract Lectin affinity chromatography was used to demonstrate that digitonin-solubilized opiate receptors contain a carbohydrate moiety. Receptors solubilized from toad, rat, chicken, bovine and human brains were retained on columns of wheat germ agglutinin (WGA)-agarose and eluted specifically with N-acetylglucosamine. The fraction retained and subsequently eluted ranged from 40–60% of the applied receptors. The eluted receptor was enriched approx. 30-fold. Evidence is presented which shows that the site of lectin interaction is functionally independent of the opiate binding site.

Radioautography of binding of tritiated diprenorphine to opiate receptors in the rat

Abstract Radioautography of tritiated diprenorphine in rat brain indicates anatomic distribution of receptors with a greater degree of precision than is possible using dissection techniques. The results of this study largely confirm those of others but indicate some differences in receptor distribution in the thalamus. Differential receptor binding in the periaquaductal gray matter with the highest counts lying laterally is an original observation.

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.

Metabolism of polyamines in mouse neuroblastoma cells in culture. Formation of gaba and putreanine.

—Polyamine metabolism of mouse neuroblastoma cells grown in culture was studied with special reference to the synthesis of GABA from putrescine and putreanine from spermidine. This study shows that neuroblastoma cells in the presence of a complete culture medium containing calf serum readily metabolized [14C]putrescine to GABA; the rate of synthesis is similar to the rate of synthesis of spermidine from putrescine. In the absence of serum the conversion of putrescine to GABA is minimal. In the presence of serum GABA formation is completely inhibited by the diamine oxidase inhibitor aminoguanidine. GABA synthesis does not occur in the absence of cells. The GABA synthesized is not readily metabolized to succinate or homocarnosine. Mouse neuroblastoma cells metabolized [14C]ornithine to putrescine, GABA, and spermidine. Spermidine was metabolized to putrescine, putreanine and spermine.

The nature of opiate receptors in toad brain

Abstract We have previously reported that particulate fractions from the brain of Bufo marinus bind enkephalins and their analogues very poorly, suggesting a low level or virtual absence of δ receptors. A detailed examination has revealed that the predominant opiate receptor subclass in toad brain is of the benzomorphan preferring type. This subclass, though similar to the κ and σ binding sites found in mammalian brain, seems to have a higher affinity for μ-preferring ligands than its mammalian counterpart. Based in our findings, we estimate that toad brain contains 60–70% κ/σ sites, 20–30% μ sites and very few if any δ sites.

Effects of chronic food restriction on mu and kappa opioid binding in rat forebrain: a quantitative autoradiographic study

It was previously observed that chronic food restriction lowers the threshold for lateral hypothalamic self-stimulation in a manner that is reversible by mu- and kappa-selective opioid antagonists. The present quantitative autoradiographic study was designed to investigate whether chronic food restriction alters regional mu and kappa opioid binding in brain. [3H]DAGO (mu) and mu/delta blocked [3H]BMZ (kappa) binding were analyzed in 34 brain regions from the medial prefrontal cortex to posterior hypothalamus. Significant reductions in mu binding were observed in caudal portions of the medial and lateral habenula, and the basolateral and basomedial nuclei of the amygdala. kappa binding was similarly reduced in medial habenula. Large increases in kappa binding were observed in the bed nucleus of the stria terminalis, ventral pallidum, and medial preoptic area. The possible involvement of these changes in the sensitization of reward by food restriction is discussed.

Immunohistochemical localization of MU-opioid receptors in rat brain using antibodies generated against a peptide sequence present in a purified MU-opioid binding protein

Light-microscope visualization in rat brain of a pattern of distribution of immunoreactivity, which included immunolabeled perikarya and beaded processes, was achieved using an immunoaffinity purified polyclonal antibody, Ab165, which recognizes the amino acid sequence, IRNLRQDRSKYY, found in the mu-opioid binding protein purified in our laboratory. Immunohistochemical staining with Ab165 was carried out by the avidin-biotin procedure. Antibody, preabsorbed with antigen, served as control. Extensive immunoreactivity was seen in the hippocampal formation, the amygdaloid complex, the striatal complex, cortical regions, select areas of the thalamus and hypothalamus and in laminae I and II of the dorsal horn in spinal cord. The distribution of immunoreactivity in the rat brain of antibody 165, which recognizes a purified mu-opioid binding protein, is concordant with the distribution of mu-opioid binding sites as determined by other laboratories in autoradiographic, electrophysiological and immunocytochemical studies. These findings have enabled us to distinguish areas possessing large fields of mu-opioid receptor containing cell bodies from areas possessing dense networks of immunolabeled neuronal processes or mixtures of both.

Effects of stress and β-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.