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Background and Purpose: Chronic morphine administration produces tolerance in vivo and attenuation of mu opioid receptor (MOR)-mediated G-protein activation measured in vitro, but the relationship between these adaptations is not clear. The present study examined MOR-mediated G-protein activation in the CNS of mice with different levels of morphine tolerance.Experimental Approach: Mice were implanted with morphine pellets, with or without supplemental morphine injections, to induce differing levels of tolerance as determined by a range of MOR-mediated behaviours. MOR function was measured using agonist-stimulated [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) and receptor binding throughout the CNS.Key Results: Morphine pellet implantation produced 6-12-fold tolerance in antinociceptive assays, hypothermia and Straub tail, as measured by the ratio of morphine ED(50) values between morphine-treated and control groups. Pellet implantation plus supplemental injections produced 25-50-fold tolerance in these tests. In morphine pellet-implanted mice, MOR-stimulated [(35)S]GTPgammaS binding was significantly reduced only in the nucleus tractus solitarius (NTS) and spinal cord dorsal horn in tissue sections from morphine pellet-implanted mice. In contrast, MOR-stimulated [(35)S]GTPgammaS binding was significantly decreased in most regions examined in morphine pellet+morphine injected mice, including nucleus accumbens, caudate-putamen, periaqueductal gray, parabrachial nucleus, NTS and spinal cord.Conclusions and Implications: Tolerance and the regional pattern of apparent MOR desensitization were influenced positively by the level of morphine exposure. These results indicate that desensitization of MOR-mediated G-protein activity is more regionally widespread upon induction of high levels of tolerance, suggesting that this response contributes more to high than low levels of tolerance to CNS-mediated effects of morphine. [ABSTRACT FROM AUTHOR]

Exposure to particulate matter (PM), a major component of air pollution, is associated with exacerbation of chronic respiratory disease, and infectious diseases such as community-acquired pneumonia. Although PM can cause adverse health effects through direct damage to host cells, our previous study showed that PM can also impact bacterial behaviour by promoting in vivo colonization. In this study we describe the genetic mechanisms involved in the bacterial response to exposure to black carbon (BC), a constituent of PM found in most sources of air pollution. We show that Staphylococcus aureus strain USA300 LAC grown in BC prior to inoculation showed increased murine respiratory tract colonization and pulmonary invasion in vivo, as well as adhesion and invasion of human epithelial cells in vitro. Global transcriptional analysis showed that BC has a widespread effect on S. aureus transcriptional responses, altering the regulation of the major virulence gene regulators Sae and Agr and causing increased expression of genes encoding toxins, proteases and immune evasion factors. Together these data describe a previously unrecognized causative mechanism of air pollution-associated infection, in that exposure to BC can increase bacterial colonization and virulence factor expression by acting directly on the bacterium rather than via the host., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Implementation of regulatory standards has reduced exhaust emissions of particulate matter from road traffic substantially in the developed world. However, nonexhaust particle emissions arising from the wear of brakes, tires, and the road surface, together with the resuspension of road dust, are unregulated and exceed exhaust emissions in many jurisdictions. While knowledge of the sources of nonexhaust particles is fairly good, source-specific measurements of airborne concentrations are few, and studies of the toxicology and epidemiology do not give a clear picture of the health risk posed. This paper reviews the current state of knowledge, with a strong focus on health-related research, highlighting areas where further research is an essential prerequisite for developing focused policy responses to nonexhaust particles.

Background: Inflammation, oxidative stress and reduced cardiopulmonary function following exposure to ultrafine particles (UFP) from airports has been reported but the biological pathways underlying these toxicological endpoints remain to be explored. Urinary metabolomics offers a robust method by which changes in cellular pathway activity can be characterised following environmental exposures., Objective: We assessed the impact of short-term exposures to UFP from different sources at a major airport on the human urinary metabolome., Methods: 21 healthy, non-smoking volunteers (aged 19-27 years) were repeatedly (2-5 visits) exposed for 5h to ambient air at Amsterdam Airport Schiphol, while performing intermittent, moderate exercise. Pre- to-post exposure changes in urinary metabolite concentrations were assessed via 1 H NMR spectroscopy and related to total and source-specific particle number concentrations (PNC) using linear mixed effects models., Results: Total PNC at the exposure site was on average, 53,500 particles/cm 3 (range 10,500-173,200) and associated with significant reductions in urinary taurine (-0.262 AU, 95% CI: -0.507 to -0.020) and dimethylamine concentrations (-0.021 AU, 95% CI: -0.040 to -0.067). Aviation UFP exposure accounted for these changes, with the reductions in taurine and dimethylamine associating with UFP produced during both aircraft landing and take-off. Significant reductions in pyroglutamate concentration were also associated with aviation UFP specifically, (-0.005 AU, 95% CI: -0.010 - <0.000) again, with contributions from both landing and take-off UFP exposure. While non-aviation UFPs induced small changes to the urinary metabolome, their effects did not significantly impact the overall response to airport UFP exposure., Discussion: Following short-term exposures at a major airport, aviation-related UFP caused the greatest changes to the urinary metabolome. These were consistent with a heightened antioxidant response and altered nitric oxide synthesis. Although some of these responses could be adaptive, they appeared after short-term exposures in healthy adults. Further study is required to determine whether long-term exposures induce injurious effects., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

In December 2019, a novel disease, coronavirus disease 19 (COVID-19), emerged in Wuhan, People's Republic of China. COVID-19 is caused by a novel coronavirus (SARS-CoV-2) presumed to have jumped species from another mammal to humans. This virus has caused a rapidly spreading global pandemic. To date, over 300,000 cases of COVID-19 have been reported in England and over 40,000 patients have died. While progress has been achieved in managing this disease, the factors in addition to age that affect the severity and mortality of COVID-19 have not been clearly identified. Recent studies of COVID-19 in several countries identified links between air pollution and death rates. Here, we explored potential links between major fossil fuel-related air pollutants and SARS-CoV-2 mortality in England. We compared current SARS-CoV-2 cases and deaths from public databases to both regional and subregional air pollution data monitored at multiple sites across England. After controlling for population density, age and median income, we show positive relationships between air pollutant concentrations, particularly nitrogen oxides, and COVID-19 mortality and infectivity. Using detailed UK Biobank data, we further show that PM 2.5 was a major contributor to COVID-19 cases in England, as an increase of 1 m 3 in the long-term average of PM 2.5 was associated with a 12% increase in COVID-19 cases. The relationship between air pollution and COVID-19 withstands variations in the temporal scale of assessments (single-year vs 5-year average) and remains significant after adjusting for socioeconomic, demographic and health-related variables. We conclude that a small increase in air pollution leads to a large increase in the COVID-19 infectivity and mortality rate in England. This study provides a framework to guide both health and emissions policies in countries affected by this pandemic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Studies have emphasised the importance of combustion-derived particles in eliciting adverse health effects, especially those produced by diesel vehicles. In contrast, few investigations have explored the potential toxicity of particles derived from tyre and brake wear, despite their significant contributions to total roadside particulate mass. The objective of this study was to compare the relative toxicity of compositionally distinct brake abrasion dust (BAD) and diesel exhaust particles (DEP) in a cellular model that is relevant to human airways. Although BAD contained considerably more metals/metalloids than DEP (as determined by inductively coupled plasma mass spectrometry) similar toxicological profiles were observed in U937 monocyte-derived macrophages following 24 h exposures to 4-25 μg ml-1 doses of either particle type. Responses to the particles were characterised by dose-dependent decreases in mitochondrial depolarisation (p ≤ 0.001), increased secretion of IL-8, IL-10 and TNF-α (p ≤ 0.05 to p ≤ 0.001) and decreased phagocytosis of S. aureus (p ≤ 0.001). This phagocytic deficit recovered, and the inflammatory response resolved when challenged cells were incubated for a further 24 h in particle-free media. These responses were abrogated by metal chelation using desferroxamine. At minimally cytotoxic doses both DEP and BAD perturbed bacterial clearance and promoted inflammatory responses in U937 cells with similar potency. These data emphasise the requirement to consider contributions of abrasion particles to traffic-related clinical health effects.

Cigarette smokers with brain damage involving the insular cortex display cessation of tobacco smoking, suggesting that this region may contribute to nicotine addiction. In the present study, we speculated that molecules in the insular cortex that are sensitive to experimental traumatic brain injury (TBI) in mice might provide leads to ameliorate nicotine addiction. Using targeted lipidomics, we found that TBI elicited substantial increases of a largely uncharacterized lipid, N-acyl-glycine, N-oleoyl-glycine (OlGly), in the insular cortex of mice. We then evaluated whether intraperitoneal administration of OlGly would alter withdrawal responses in nicotine-dependent mice as well as the rewarding effects of nicotine, as assessed in the conditioned place preference paradigm (CPP). Systemic administration of OlGly reduced mecamylamine-precipitated withdrawal responses in nicotine-dependent mice and prevented nicotine CPP. However, OlGly did not affect morphine CPP, demonstrating a degree of selectivity. Our respective in vitro and in vivo observations that OlGly activated peroxisome proliferator-activated receptor alpha (PPAR-α) and the PPAR-α antagonist GW6471 prevented the OlGly-induced reduction of nicotine CPP in mice suggests that this lipid acts as a functional PPAR-α agonist to attenuate nicotine reward. These findings raise the possibility that the long chain fatty acid amide OlGly may possess efficacy in treating nicotine addiction., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Combustion of biodiesels in place of fossil diesel (FD) has been proposed as a method of reducing transport-related toxic emissions in Europe. While biodiesel exhaust (BDE) contains fewer hydrocarbons, total particulates and carbon monoxide than FD exhaust (FDE), its high nitrogen oxide and ultrafine particle content may still promote pulmonary pathophysiologies., Main Body: Using a complement of in vitro and in vivo studies, this review documents progress in our understanding of pulmonary responses to BDE exposure. Focusing initially on hypothesis-driven, targeted analyses, the merits and limitations of comparing BDE-induced responses to those caused by FDE exposure are discussed within the contexts of policy making and exploration of toxicity mechanisms. The introduction and progression of omics-led workflows are also discussed, summarising the novel insights into mechanisms of BDE-induced toxicity that they have uncovered. Finally, options for the expansion of BDE-related omics screens are explored, focusing on the mechanistic relevance of metabolomic profiling and offering rationale for expansion beyond classical models of pulmonary exposure., Conclusion: Together, these discussions suggest that molecular profiling methods have identified mechanistically informative, novel and fuel-specific signatures of pulmonary responses to biodiesel exhaust exposure that would have been difficult to detect using traditional, hypothesis driven approaches alone.

Significance: The environment can elicit biological responses such as oxidative stress (OS) and inflammation as a consequence of chemical, physical, or psychological changes. As population studies are essential for establishing these environment-organism interactions, biomarkers of OS or inflammation are critical in formulating mechanistic hypotheses. Recent Advances: By using examples of stress induced by various mechanisms, we focus on the biomarkers that have been used to assess OS and inflammation in these conditions. We discuss the difference between biomarkers that are the result of a chemical reaction (such as lipid peroxides or oxidized proteins that are a result of the reaction of molecules with reactive oxygen species) and those that represent the biological response to stress, such as the transcription factor NRF2 or inflammation and inflammatory cytokines., Critical Issues: The high-throughput and holistic approaches to biomarker discovery used extensively in large-scale molecular epidemiological exposome are also discussed in the context of human exposure to environmental stressors., Future Directions: We propose to consider the role of biomarkers as signs and to distinguish between signs that are just indicators of biological processes and proxies that one can interact with and modify the disease process. Antioxid. Redox Signal. 28, 852-872.

The transcription factor DeltaFosB accumulates and persists in brain in response to chronic stimulation. This accumulation after chronic exposure to drugs of abuse has been demonstrated previously by Western blot most dramatically in striatal regions, including dorsal striatum (caudate/putamen) and nucleus accumbens. In the present study, we used immunohistochemistry to define with greater anatomical precision the induction of DeltaFosB throughout the rodent brain after chronic drug treatment. We also extended previous research involving cocaine, morphine, and nicotine to two additional drugs of abuse, ethanol and Delta(9)-tetrahydrocannabinol (Delta(9)-THC, the active ingredient in marijuana). We show here that chronic, but not acute, administration of each of four drugs of abuse, cocaine, morphine, ethanol, and Delta(9)-THC, robustly induces DeltaFosB in nucleus accumbens, although different patterns in the core vs. shell subregions of this nucleus were apparent for the different drugs. The drugs also differed in their degree of DeltaFosB induction in dorsal striatum. In addition, all four drugs induced DeltaFosB in prefrontal cortex, with the greatest effects observed with cocaine and ethanol, and all of the drugs induced DeltaFosB to a small extent in amygdala. Furthermore, all drugs induced DeltaFosB in the hippocampus, and, with the exception of ethanol, most of this induction was seen in the dentate. Lower levels of DeltaFosB induction were seen in other brain areas in response to a particular drug treatment. These findings provide further evidence that induction of DeltaFosB in nucleus accumbens is a common action of virtually all drugs of abuse and that, beyond nucleus accumbens, each drug induces DeltaFosB in a region-specific manner in brain., ((c) 2008 Wiley-Liss, Inc.)

Activation of the cannabinoid type 1 (CB1) receptor, a major G-protein-coupled receptor in brain, acts to regulate neuronal excitability and has been shown to mediate the anticonvulsant effects of cannabinoids in several animal models of seizure, including the rat pilocarpine model of acquired epilepsy. However, the long-term effects of status epilepticus on the expression and function of the CB1 receptor have not been described. Therefore, this study was initiated to evaluate the effect of status epilepticus on CB1 receptor expression, binding, and G-protein activation in the rat pilocarpine model of acquired epilepsy. Using immunohistochemistry, we demonstrated that status epilepticus causes a unique "redistribution" of hippocampal CB1 receptors, consisting of specific decreases in CB1 immunoreactivity in the dense pyramidal cell layer neuropil and dentate gyrus inner molecular layer, and increases in staining in the CA1-3 strata oriens and radiatum. In addition, this study demonstrates that the redistribution of CB1 receptor expression results in corresponding functional changes in CB1 receptor binding and G-protein activation using [3H] R+-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl](1-napthalen-yl)methanone mesylate (WIN55,212-2) and agonist-stimulated [35S]GTPgammaS autoradiography, respectively. The redistribution of CB1 receptor-mediated [35S]GTPgammaS binding was 1) attributed to an altered maximal effect (Emax) of WIN55,212-2 to stimulate [35S]GTPgammaS binding, 2) reversed by the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), 3) confirmed by the use of other CB1 receptor agonists, and 4) not reproduced in other G-protein-coupled receptor systems examined. These results demonstrate that status epilepticus causes a unique and selective reorganization of the CB1 receptor system that persists as a permanent hippocampal neuronal plasticity change associated with the development of acquired epilepsy.

The phosphatidylinositol (PI) cascade plays a pivotal role in mediating behavioral tolerance to the antinociceptive effects of morphine. Earlier we reported that antinociceptive tolerance was completely reversed 30 min after the administration of inhibitors of each step in the PI cascade. The aim of this study was to determine whether injection of a single dose of protein kinase C (PKC) inhibitor would elicit a prolonged reversal of morphine tolerance for up to 24 h. Three days after implantation of placebo- or 75-mg morphine pellets, mice received intracerebroventricular (i.c.v.) injections of vehicle or PKC inhibitor drug. Morphine challenge doses were then administered 4, 8 and 24 h later to test for tolerance reversal. In non-tolerant mice, Gö-7874 and sangivamycin had no effect on the potency of morphine. However, Gö-7874 and sangivamycin significantly reversed morphine tolerance at 4, 8 and 24 h. In addition, the role of PKC in morphine physical dependence was determined. Gö-7874 and sangivamycin by themselves did not precipitate spontaneous morphine withdrawal. Therefore, experiments were conducted to determine whether the PKC inhibitors would block naloxone-precipitated withdrawal. However, neither a 30-min nor a 24-h pretreatment with Gö-7874 or sangivamycin blocked naloxone withdrawal. Our results along with other publications indicate that PKC is a pivotal kinase essential for maintaining animals in an opioid tolerant state. Finally, the use of persistent PKC inhibitors that lasted for 24 h demonstrated that the neuronal systems in these animals did not adapt by increasing the activity of other protein kinase cascades to re-establish morphine tolerance., (Copyright 2002 Elsevier Science B.V.)

Anterior cingulate cortex (ACC) has a role in pain processing, however, little is known about opioid system organization and actions. This rodent study defines opioid architecture in the perigenual and midcingulate divisions of ACC, relates mu-opioid receptor binding and G-protein activation, and localizes such binding to afferent axons with knife-cut lesions and specifically to noradrenergic terminals with immunotoxin lesions (anti-dopamine beta-hydroxylase-saporin; anti-DBH-saporin). [(3)H]Tyr-D-AlaGly-MePhe-Gly-ol (DAMGO) binding was highest in perigenual areas 32 and 24 with a peak in layer I. Midcingulate area 24' and posterior cingulate area 29 had overall lower binding in each layer. In contrast, DAMGO-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding in area 24' was similar to that in area 24, whereas area 29 had low and homogeneous binding. Undercut lesions reduced [(3)H]DAMGO binding in all layers with the greatest loss in layer I (-65%), whereas DAMGO-stimulated [(35)S]GTPgammaS binding losses occurred in only layers I-III. Anti-DBH-saporin reduced [(3)H]DAMGO binding in layer I of area 24; DAMGO-stimulated [(35)S]GTPgammaS binding was unchanged in areas 24' and 29. Correlation analysis of receptor and G-protein activation before and after undercut lesions suggested there were a greater number of DAMGO receptor sites for each G-protein on axons, than on somata and proximal dendrites. Finally, perigenual and midcingulate cortices have different opioid architectures due to a higher proportion of mu-opioid receptors expressed by afferent axons in areas 24 and 32.

The subregional distribution of mu opioid receptors and corresponding G-protein activation were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. The topography of mu binding sites was defined using autoradiography with [(3)H]DAMGO, a selective mu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identified with DAMGO-stimulated guanylyl 5'(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Within the striatum, the distribution of [(3)H]DAMGO binding sites was characterized by a distinct dorsal-ventral gradient with a higher concentration of binding sites at more rostral levels of the striatum. [(3)H]DAMGO binding was further distinguished by the presence of patch-like aggregations within the caudate, as well as smaller areas of very dense receptor binding sites, previously identified in human striatum as neurochemically unique domains of the accumbens and putamen (NUDAPs). The amygdala contained the highest concentration of [(3)H]DAMGO binding sites measured in this study, with the densest levels of binding noted within the basal, accessory basal, paralaminar, and medial nuclei. In the striatum and amygdala, the distribution of DAMGO-stimulated G-protein activation largely corresponded with the distribution of mu binding sites. The central and medial nuclei of the amygdala, however, were notable exceptions. Whereas the concentration of [(3)H]DAMGO binding sites in the central nucleus of the amygdala was very low, the concentration of DAMGO-stimulated G-protein activation in this nucleus, as measured with [(35)S]GTPgammaS binding, was relatively high compared to other portions of the amygdala containing much higher concentrations of [(3)H]DAMGO binding sites. The converse was true in the medial nucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [(3)H]DAMGO and [(35)S]GTPgammaS binding within the amygdala, particularly the medial nucleus, formed a continuum with the substantia innominata and bed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates., (Copyright 2001 Wiley-Liss, Inc.)

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G-protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [(35)S]guanosine-5'-(gamma-O-thio) triphosphate ([(35)S]GTPgammaS) binding by the high efficacy agonist WIN 55,212-2 was reduced by only 20-25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and (9)-tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212-2 were generally unchanged. Saturation analysis of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding showed that decreased stimulation by WIN 55,212-2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net-stimulated [(35)S]GTPgammaS binding. The apparent maximal number of binding sites (B(max)) values of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net-stimulated [(35)S]GTPgammaS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region-dependent increases in the apparent efficiency of CB1 receptor-mediated G-protein activation in heterozygous CB1 knockout mice.

In previous studies from our laboratory, chronic noncontingent morphine administration decreased mu opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, mu opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg. kg(-1). d(-1). mu opioid-stimulated [(35)S]GTPgammaS and [(3)H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [(35)S]GTPgammaS autoradiography also examined other G-protein-coupled receptors, including delta opioid, ORL-1, GABA(B), adenosine A(1), cannabinoid, and 5-HT(1A). In brains from heroin self-administering rats, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in mu-activated G-proteins, [(3)H]naloxone binding demonstrated increased mu opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in mu receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and mu receptor binding. No effects on agonist-stimulated [(35)S]GTPgammaS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease mu-stimulated [(35)S]GTPgammaS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.

The sulfhydryl alkylating reagent N-ethylmaleimide (NEM) blocks opioid receptor binding and receptor/G-protein coupling. Sodium partially restores [(3)H]naloxone binding after inhibition by NEM to reveal sodium-dependent [(3)H]naloxone sites, defined as binding in the presence of 50-100 mM NaCl after treatment of membranes or sections with 750 microM NEM. In the present study, receptor autoradiography of [(3)H]naloxone binding in control and NEM-treated tissue was used to examine the anatomical distribution of sodium-dependent [(3)H]naloxone sites in rat brain. In brain membranes, the pharmacology of sodium-dependent [(3)H]naloxone sites was consistent with that of mu opioid receptors. Relatively high IC(50) values for agonists and lack of effect of Gpp(NH)p on DAMGO displacement of [(3)H]naloxone binding in NEM-treated membranes indicated that the sodium-dependent sites were low affinity sites, presumably uncoupled from G-proteins. Autoradiograms revealed that NEM treatment dramatically reduced [(3)H]naloxone binding in all brain regions. However, [(3)H]naloxone binding was increased in specific regions in NEM-treated sections in the presence of sodium, including bed nucleus of the stria terminalis, interpeduncular nucleus, periaqueductal gray, parabrachial nucleus, locus coeruleus, and commissural nucleus tractus solitarius. Sodium-dependent [(3)H]naloxone binding sites were not found in other areas that exhibited [(3)H]naloxone binding in control tissue, including the striatum and thalamus. These studies revealed the presence of a subpopulation of [(3)H]naloxone binding sites which are sodium-dependent and have a unique regional distribution in the rat brain., (Copyright 2000 Wiley-Liss, Inc.)

Coupling of receptors to G-proteins can be assessed by the ability of specific agonists to stimulate [35S]GTPgammaS binding in both brain membranes and sections in the presence of excess GDP. In some brain regions, however, high basal activity makes it difficult to detect agonist-stimulated [35S]GTPgammaS binding. The present study suggests a modification of the assay to reduce basal [35S]GTPgammaS binding and thus increase the signal:noise ratio. Adenosine A1 receptors belong to the class of G-protein-coupled receptors that activate Gi/Go proteins in brain. In the present study, the A1 agonist R(-)N6-(2-phenylisopropyl)adenosine (R-PIA) stimulated [35S]GTPgammaS binding in brain regions known to contain A1 receptors, including cerebellum, hippocampus and dentate gyrus, medial geniculate body, superior colliculus, certain thalamic nuclei, cerebral cortex, piriform cortex, caudate-putamen, and nucleus accumbens. Treatment of sections and membranes with adenosine deaminase (ADase), which is typically used in adenosine assays to eliminate endogenous adenosine, reduced basal [35S]GTPgammaS binding. In addition, for cannabinoid and mu-opioid agonists, the percent stimulation of [35S]GTPgammaS binding was approximately doubled when ADase was included in the assay. These results suggest that endogenous adenosine contributes significantly to basal [35S]GTPgammaS binding in certain brain regions, and that this activity may be reduced by the addition of ADase, thus improving the signal:noise ratio of agonist-stimulated [35S]GTPgammaS binding.

Chronic treatment of rats with delta9-tetrahydrocannabinol (delta9-THC) results in tolerance to its acute behavioral effects. In a previous study, 21-day delta9-THC treatment in rats decreased cannabinoid activation of G proteins in brain, as measured by in vitro autoradiography of guanosine-5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding. The present study investigated the time course of changes in cannabinoid-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding in both brain sections and membranes, following daily delta9-THC treatments for 3, 7, 14, and 21 days. Autoradiographic results showed time-dependent decreases in WIN 55212-2-stimulated [35S]GTPgammaS and [3H]WIN 55212-2 binding in cerebellum, hippocampus, caudate-putamen, and globus pallidus, with regional differences in the rate and magnitude of down-regulation and desensitization. Membrane binding assays in these regions showed qualitatively similar decreases in WIN 55212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding (using [3H]SR141716A), and demonstrated that decreases in ligand binding were due to decreases in maximal binding values, and not ligand affinities. These results demonstrated that chronic exposure to delta9-THC produced time-dependent and region-specific down-regulation and desensitization of brain cannabinoid receptors, which may represent underlying biochemical mechanisms of tolerance to cannabinoids.

Agonist-stimulated [35S]GTPgammaS binding allows the visualization of receptor-activated G-proteins, thus revealing the anatomical localization of functional receptor activity. In the present study, agonist-stimulated [35S]GTPgammaS binding was used to demonstrate mu and kappa1 opioid-stimulated [35S]GTPgammaS binding in tissue sections and membranes from cynomolgus monkey brain using DAMGO and U50,488H, respectively. Concentrations of agonists required to produce maximal stimulation of [35S]GTPgammaS binding were determined in membranes from the frontal poles of the brain. Receptor specificity was verified in both membranes and sections by inhibiting agonist-stimulated [35S]GTPgammaS binding with the appropriate antagonist. Mu opioid-stimulated [35S]GTPgammaS binding was high in areas including the amygdala, ventral striatum, caudate, putamen, medial thalamus and hypothalamus. Dense mu-stimulated [35S]GTPgammaS binding was also found in brainstem nuclei including the interpeduncular nucleus, parabrachial nucleus and nucleus of the solitary tract. Kappa1 opioid-stimulated [35S]GTPgammaS binding was high in limbic and association cortex, ventral striatum, caudate, putamen, globus pallidus, claustrum, amygdala, hypothalamus and substantia nigra. These results demonstrate the applicability of [35S]GTPgammaS autoradiography to examine receptor-activated G-proteins in the primate brain and reveal functional mu and kappa1 opioid receptor activity that may contribute to the reported central nervous system effects of opiates.