High and low doses of cocaine intake are differentially regulated by dopamine D2 receptors in the ventral tegmental area and the nucleus accumbens.

Dopamine D2 receptors (D2Rs) in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) are associated with vulnerability to addiction; however, whether D2Rs in these two brain regions play differential roles in regulation of drug intake is unknown. Here, we compared the effect of decreased mRNA level of Drd2 in each region on cocaine self-administration in a dose-response function. Drd2 mRNA levels in rat VTA or NAc were knocked down by bilateral microinjection of lentivirus coding shRNAs against rat Drd2 or scrambled shRNA. Drd2 knockdown was persistent and stable between 20 and 90 days after lentiviral infection. Animals were trained to self-administer cocaine 20 days after Drd2 shRNA treatment. Compared to scrambled shRNA treated rats, Drd2 knockdown in the VTA increased cocaine self-administration at all tested doses (0.02-0.56 mg/kg/infusion) producing an upward shift (both the ascending and descending limb) in the dose-response curve of cocaine self-administration. In contrast, intra-NAc knockdown increased cocaine self-administration only on the ascending limb of the dose-response curve (0.02-0.07 mg/kg/infusion). These data suggest that D2Rs in the VTA, not in the NAc, regulate high-dose cocaine intake. The present study not only demonstrates that low levels of D2Rs in either region increase low doses of cocaine intake, but also reveals for the first time their dissociable roles in limiting high doses of cocaine self-administration.

Regional differences in mu and kappa opioid receptor G-protein activation in brain in male and female prairie voles.

Prairie voles are unusual mammals in that, like humans, they are capable of forming socially monogamous pair bonds, display biparental care, and engage in alloparental behaviors. Both mu and kappa opioid receptors are involved in behaviors that either establish and maintain, or result from pair bond formation in these animals. Mu and kappa opioid receptors both utilize inhibitory G-proteins in signal transduction mechanisms, however the efficacy by which these receptor subtypes stimulate G-protein signaling across the prairie vole neuraxis is not known. Utilizing [(35)S]GTPγS autoradiography, we characterized the efficacy of G-protein stimulation in coronal sections throughout male and female prairie vole brains by [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin (DAMGO) and U50,488H, selective mu and kappa opioid agonists, respectively. DAMGO stimulation was highest in the forebrain, similar to that found with other rodent species. U-50,488H produced greater stimulation in prairie voles than is typically seen in mice and rats, particularly in select forebrain areas. DAMGO produced higher stimulation in the core versus the shell of the nucleus accumbens (NAc) in females, while the distribution of U-50,488H stimulation was the opposite. There were no gender differences for U50,488H stimulation of G-protein activity across the regions examined, while DAMGO stimulation was greater in sections from females compared to those from males for NAc core, entopeduncular nucleus, and hippocampus. These data suggest that the kappa opioid system may be more sensitive to manipulation in prairie voles compared to mice and rats, and that female prairie voles may be more sensitive to mu agonists in select brain regions than males.

Colocalization of mu-opioid receptors and activated G-proteins in rat cingulate cortex.

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.

mu Opioid receptor-mediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6-monoacetylmorphine compared with morphine.

The efficacy of heroin metabolites for the stimulation of mu opioid receptor-mediated G-protein activation was investigated using agonist-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate binding. In rat thalamic membranes, heroin and its primary metabolite, 6-monoacetylmorphine (6-MAM), were more efficacious than morphine or morphine-6-beta D-glucuronide. This increased efficacy was not due to increased action of heroin and 6-MAM at delta receptors, as determined by competitive antagonism by naloxone, lack of antagonism by naltrindole, and competitive partial antagonism with morphine. In agreement with this interpretation, the same relative efficacy profile of heroin and its metabolites was observed at the cloned human mu opioid receptor expressed in C6 glioma cells. Moreover, these efficacy differences were GDP-dependent in a manner consistent with accepted mechanisms of receptor-mediated G-protein activation. The activity of heroin was attributed to in vitro deacetylation to 6-MAM, as confirmed by HPLC analysis. These results indicate that the heroin metabolite 6-MAM possesses higher efficacy than other heroin metabolites at mu opioid receptors, which may contribute to the higher efficacy of heroin compared with morphine in certain behavioral paradigms in vivo.

Synthesis of 2beta-acyl-3beta-(substituted naphthyl)-8-azabicyclo[3.2.1]octanes and their binding affinities at dopamine and serotonin transport sites.

A series of 3beta-naphthyltropane derivatives were synthesized and found to show high affinity at both the dopamine and serotonin transporter sites, leading to some of the most potent inhibitors known based on the tropane structure. Comparative molecular field analysis (CoMFA) models were developed for both dopamine and serotonin transporter binding data. These models provide insights into those factors that influence binding at the two transporters.

Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate.

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.

Synthesis and monoamine transporter affinity of 3beta-(4-(2-pyrrolyl)phenyl)-8-azabicycl.

3Beta-(5-indolyl)-8-azabicyclo[3.2.1]octanes display potent binding affinity for both the dopamine and serotonin transporters, while certain 3beta-(4-(2-pyrrolyl)phenyl)-8-azabicyclo[3.2.1]octanes selectively bind to the serotonin transporter.

Chronic intrathecal morphine administration produces homologous mu receptor/G-protein desensitization specifically in spinal cord.

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.

Cannabinoid agonist signal transduction in rat brain: comparison of cannabinoid agonists in receptor binding, G-protein activation, and adenylyl cyclase inhibition.

To investigate differences in agonist affinity, potency, and efficacy across rat brain regions, five representative cannabinoid compounds were investigated in membranes from three different rat brain regions for their ability to maximally stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding and bind to cannabinoid receptors (measured by inhibition of [(3)H]antagonist binding) under identical assay conditions. In all three brain regions, the rank order of potency for the stimulation of [(35)S]GTPgammaS binding and the inhibition of [(3)H]SR141716A binding for these compounds were identical, with CP55940 approximately levonantradol > WIN55212-2 >/= Delta(9)-tetrahydrocannabinol (Delta(9)-THC) > methanandamide. The rank order of efficacy was not related to potency, and relative maximal agonist effects varied across regions. Receptor binding fit to a three-site model for most agonists, stimulation of [(35)S]GTPgammaS binding fit to a two-site model for all agonists, and high-affinity receptor binding did not appear to produce any stimulation of [(35)S]GTPgammaS binding. WIN55212-2, methanandamide, and Delta(9)-THC also were assayed for the inhibition of adenylyl cyclase in cerebellar membranes. The rank orders of potency and efficacy were similar to those for [(35)S]GTPgammaS binding, but the efficacies and potencies of methanandamide and Delta(9)-THC compared with WIN55212-2 were higher for adenylyl cyclase inhibition, implying receptor/G-protein reserve.

Fluorine-18-labeled tropane analogs for PET imaging studies of the dopamine transporter.

A series of PET imaging studies were conducted with two fluorine-18-labeled tropane analoges, [(18)F](+)-FTT and [(18)F](+)-FCT. Both compounds possessed a high affinity and selectivity for the dopamine transporter and had a higher accumulation in the basal ganglia, a brain region having a high density of the dopamine transporter (DAT) than the cerebellum, a reference region devoid of dopaminergic terminals. [(18)F](+)-FCT had a higher brain uptake and more suitable basal ganglia:cerebellum (BG:Cb) ratio than [(18)F](+)-FTT. [(18)F](+)-FCT also displayed reversible binding kinetics in vivo, indicating that the measurement of DAT density in vivo with PET will be relatively insensitive to changes in cerebral blood flow that can occur as a consequence of disease or prolonged cocaine abuse. The uptake of [(18)F](+)-FCT was also displaced by an intravenous injection of cocaine (1.0 mg/kg), which is consistent with the labeling of the DAT in vivo by this radiotracer. These data suggest that [(18)F](+)-FCT may be a suitable radiotracer for studying DAT function in vivo with PET.

Effects of sodium on agonist efficacy for G-protein activation in mu-opioid receptor-transfected CHO cells and rat thalamus.

1. Sodium ions inhibit spontaneous G(i)/G(o)-coupled receptor activity and promote agonist-induced responses in vitro. The effects of sodium on the relative efficacy of opioid agonists for G-protein activation was measured by guanosine-5'-O-(gamma-(35)S)-triphosphate ([(35)S]-GTPgammaS) binding in membranes from two mu-opioid receptor-containing systems: CHO cells stably transfected with mouse mureceptors (mMOR-CHO cells) and rat thalamus. 2. NaCl inhibited basal [(35)S]-GTPgammaS binding in both systems, and this effect was partially mimicked by KCl. In mMOR-CHO membranes, net [(35)S]-GTPgammaS binding stimulated by partial but not full agonists was inhibited by NaCl with a potency that was inversely proportional to agonist efficacy. Monovalent cations were required for agonist-stimulated [(35)S]-GTPgammaS binding in this system, and increasing NaCl concentrations magnified relative efficacy differences among agonists. 3. In thalamic membranes, which contain a lower receptor:G-protein ratio than mMOR-CHO cells, similar monovalent cation effects were observed, with two exceptions: (1) [(35)S]-GTPgammaS binding stimulated by both full and partial agonists was inhibited by NaCl; and (2) monovalent cations were not required to observe agonist-stimulated [(35)S]-GTPgammaS binding. 4. Basal [(35)S]-GTPgammaS binding stimulated by the absence of monovalent cations resembled that of agonist-stimulated binding and was blocked by pretreatment of mMOR-CHO cells with pertussis toxin. 5. These results indicate that sodium inhibits spontaneous and agonist-occupied mu receptor-mediated G-protein activation in a manner inversely proportional to the efficacy of the agonist, and that spontaneous mu receptor activity and the relative efficacy of partial agonists acting at these receptors are both increased by increases in the stoichiometric ratio of receptors:G-proteins.

Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain.

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.

Characterization of a tropane radioligand, [(3)H]2beta-propanoyl-3beta-(4-tolyl) tropane ([(3)H]PTT), for dopamine transport sites in rat brain.

PTT (2beta-propanoyl-3beta-[4-tolyl] tropane) is a tropane analog relatively selective for dopamine transporters in binding and uptake assays in vitro, with long-acting psychostimulant properties in vivo. To explore its utility in binding to dopamine transporters, [(3)H]PTT was synthesized and assayed for binding in rat striatal membranes and by in vitro autoradiography. In membranes, binding of [(3)H]PTT was saturable to a single class of binding sites with a K(D) value of 3 nM. The pharmacology of [(3)H]PTT binding in striatal membranes was consistent with that of a ligand selective for dopamine transporters, with dopamine-selective compounds being significantly more potent in displacing [(3)H]PTT binding than those for 5-HT or norepinephrine transporters. Although the ability of various transporter inhibitors to displace both [(125)I]RTI-55 and [(3)H]PTT binding correlated significantly with each other, there was a better correlation of inhibitor potencies versus [(3)H]PTT binding and dopamine uptake than versus [(125)I]RTI-55 binding and dopamine uptake. The differences in correlations were most noticeable for compounds relatively selective at the 5-hydroxytryptamine (serotonin) transporter. The autoradiographic distribution of [(3)H]PTT binding in coronal sections was consistent with the known distribution of the dopamine transporter, with high levels of binding evident in caudate nucleus, nucleus accumbens, and olfactory tubercle. Moderate densities of [(3)H]PTT binding were also observed in substantia nigra pars compacta, and ventral tegmental area, as well as in the anterior cingulate cortex and portions of the hypothalamus. In addition, nonspecific binding was less than 5% of total binding. Thus, [(3)H]PTT provides an accurate and convenient marker for the dopamine transporter.

Activation of cannabinoid receptors in rat brain by WIN 55212-2 produces coupling to multiple G protein alpha-subunits with different potencies.

Previous studies had shown that the amplification factors for cannabinoid receptors, defined as the number of total G proteins activated per occupied receptor, differs between several rat brain regions. In this study, we sought to determine which specific Gi/Go(alpha) subunits were activated by CB1 receptors in several rat brain regions and if this coupling might explain the regional differences in receptor/G protein amplification factors. Furthermore, we examined whether cannabinoid agonists might activate different subtypes of G(alpha) subunits with varying degrees of efficacy and/or potency. Activation of specific G proteins by cannabinoid receptors was evaluated by the ability of the agonist WIN 55212-2 to stimulate incorporation of [alpha-(32)P]azidoanilido-GTP into G(alpha) subunits in membranes. Photolabeled G proteins were either directly resolved using urea/SDS-polyacrylamide gel electrophoresis or first immunoprecipitated with specific antisera for different G(alpha) subunits before electrophoresis. Individual G(alpha) subunits were separated into distinct bands on a single gel and the amount of agonist-induced increase in radioactivity was quantified by densitometry. Stimulation of CB1 receptors by WIN 55212-2 resulted in the activation of a distinct pattern of at least five different G(ialpha)/G(oalpha) subunits in several brain regions. Furthermore, although the pattern of G proteins activated by WIN 55212-2 appeared to be similar across brain regions, slight differences were observed in both the percentage of increase and the amount of the individual G(alpha) subunits activated. Most importantly, the amount of WIN 55212-2 required to half-maximally activate individual G proteins in the cerebellum varied over a 30-fold range for different G(alpha) subunits. These results suggest that cannabinoid receptors activate multiple G proteins simultaneously in several brain regions and both the efficacy and potency of cannabinoid agonists to activate individual G(alpha) subunits may vary considerably.

Sigma-2 receptors as a biomarker of proliferation in solid tumours.

Over the past several years, our group has provided considerable evidence that the expression of sigma-2 (sigma2) receptors may serve as a biomarker of tumour cell proliferation. In these in vitro studies, sigma2 receptors were expressed 8-10 times more in proliferative (P) tumour cells than in quiescent (Q) tumour cells, and the extent and kinetics of their expression were independent of a number of biological, physiological and environmental factors often found in solid tumours. Moreover, the expression of sigma2 receptors followed both the population growth kinetics when Q-cells were recruited into the P-cell compartment and the proliferative status of human breast tumour cells treated with cytostatic concentrations of tamoxifen. However, these in vitro studies may or may not be indicative of what might occur in solid tumours. In the present study, the sigma2 receptor P:Q ratio was determined for the cells from subcutaneous 66 (diploid) and 67 (aneuploid) tumours grown in female nude mice. The sigma2 receptor P:Q ratio of the 66 tumours was 10.6 compared to the sigma2 receptor P:Q ratio of 9.5 measured for the 66 tissue culture model. The sigma2 receptor P:Q ratio of the 67 tumours was 4.5 compared to the sigma2 receptor P:Q ratio of approximately equal 8 measured for the 67 tissue culture model. The agreement between the solid tumour and tissue culture data indicates that: (1) the expression of sigma2 receptors may be a reliable biomarker of the proliferative status of solid tumours and (2) radioligands with both high affinity and high selectivity for sigma2 receptors may have the potential to non-invasively assess the proliferative status of human solid tumours using imaging techniques such as positron emission tomography or single-photon emission computerized tomography.

Region-specific changes in 5-HT(1A) receptor-activated G-proteins in rat brain following chronic buspirone.

5-Hydroxytryptamine(1A) (5-HT(1A)) receptors, which activate inhibitory G-proteins, are implicated in psychiatric disorders including anxiety and depression. Studies suggest that chronic 5-HT(1A) receptor agonist administration alters 5-HT(1A) receptor function, but the effect of chronic treatment on 5-HT(1A) receptor-activated G-proteins is unclear. In this study, agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding was examined following chronic administration of buspirone. Brains were processed for [35S]GTPgammaS autoradiography using R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) for 5-HT(1A) receptors or baclofen for GABA(B) receptors. Net 8-OH-DPAT-stimulated [35S]GTPgammaS binding was decreased by 25-30% in the septum and dorsal raphe nucleus of buspirone-treated animals. No significant changes in 8-OH-DPAT-stimulated [35S]GTPgammaS binding were found in the prefrontal, entorhinal or cingulate cortices or hippocampus in buspirone-treated rats. GABA(B) receptor-stimulated [35S]GTPgammaS binding was increased by 25% in the hippocampus, with no significant changes in any other region examined. These results demonstrate region-specific alterations in 5-HT(1A) and GABA(B) receptor-activated G-proteins following chronic buspirone treatment, which may contribute to the clinical effects of this drug.

Agonist-stimulated [35S]GTPgammaS binding in brain modulation by endogenous adenosine.

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.

Anatomical distribution of sodium-dependent [(3)H]naloxone binding sites in rat brain.

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.

Relationship of mu opioid receptor binding to activation of G-proteins in specific rat brain regions.

This study investigated the relationship between mu receptor binding and mu agonist activation of G-proteins in the rat brain. To directly compare agonist potencies in receptor binding (K(i) values) and G-protein activation (K(s) values), both agonist-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) and [(3)H]naloxone binding assays were conducted under identical conditions, using the full mu agonist [d-Ala(2), N-Me(4), Gly(5)-ol]-enkephalin (DAMGO). DAMGO exhibited biphasic competition of [(3)H]naloxone binding and stimulation of [(35)S]GTPgammaS binding in most regions. Whereas the high-affinity component represented a large percentage (50-80%) of total receptor sites, the high-affinity component of DAMGO-stimulated [(35)S]GTPgammaS binding was much lower, <30% of the total, and in most regions significant stimulation of [(35)S]GTPgammaS binding did not occur until the high-affinity binding sites were completely occupied. Moreover, the low-affinity potencies for DAMGO in receptor binding and G-protein activation were the same across different regions. Receptor-transducer amplification factors were calculated by the ratio of the apparent B(max) of net agonist-stimulated [(35)S]GTPgammaS binding to the B(max) of receptor binding. Amplification factors for the nine regions examined were relatively high and varied significantly across regions, from a ratio of 8 in the thalamus to 38 in the cortex, suggesting that the efficiency of mu opioid receptor coupling to G-proteins varies across brain regions.

Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and cannabinoid receptor-activated G proteins in rat brain.

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.