Kappa opioid receptors mediate an initial aversive component of paclitaxel-induced neuropathy.

RATIONALE: Cancer patients receiving the antineoplastic drug paclitaxel report higher incidences and longer duration of treatment-resistant depression than patients receiving other classes of chemotherapeutics. Rodents treated with paclitaxel exhibit a suite of changes in affect-like behaviors. Further, paclitaxel causes chemotherapy-induced peripheral neuropathy (CIPN) in humans and rodents. Kappa opioid receptors (KOR) have a well-established role in depression and neuropathy. The contributions of KOR signaling to paclitaxel-induced aversive-like state and CIPN in rodents remain to be explored. OBJECTIVES: We aimed to investigate whether dysregulation of the KOR/dynorphin system is associated with paclitaxel-mediated pain-like behavior and depression-like behavior. METHODS: Cancer-free male C57BL/6J mice were treated with four injections of vehicle or paclitaxel (32 mg/kg cumulative). The effects of the selective KOR antagonist norbinaltorphimine (norBNI) on paclitaxel-induced sucrose preference deficits and mechanical hypersensitivity were measured. Prodynorphin mRNA and receptor-mediated G protein activation were measured at two time points following the last paclitaxel injection using quantitative real-time polymerase chain reaction and agonist-stimulated [35S]guanosine-5'-O'-(γ-thio)-triphosphate ([35S]GTPγS) binding, respectively, in the nucleus accumbens (NAc), caudate-putamen, amygdala, and spinal cord. RESULTS: Paclitaxel produced a norBNI-reversible sucrose preference deficit, whereas mechanical hypersensitivity was not reversed by norBNI. Paclitaxel treatment increased the levels of mRNA for prodynorphin, a precursor for endogenous KOR agonists, in the NAc. Paclitaxel also had time-dependent effects on KOR-mediated G protein activation in the NAc. CONCLUSIONS: These results suggest that KOR signaling mediates an initial aversive component of paclitaxel, but not necessarily paclitaxel-induced mechanical hypersensitivity.

Stratification of Cannabinoid 1 Receptor (CB1R) Agonist Efficacy: Manipulation of CB1R Density through Use of Transgenic Mice Reveals Congruence between In Vivo and In Vitro Assays.

Synthetic cannabinoids (SCs) are an emerging class of abused drugs that differ from each other and the phytocannabinoid ∆9-tetrahydrocannabinol (THC) in their safety and cannabinoid-1 receptor (CB1R) pharmacology. As efficacy represents a critical parameter to understanding drug action, the present study investigated this metric by assessing in vivo and in vitro actions of THC, two well-characterized SCs (WIN55,212-2 and CP55,940), and three abused SCs (JWH-073, CP47,497, and A-834,735-D) in CB1 (+/+), (+/-), and (-/-) mice. All drugs produced maximal cannabimimetic in vivo effects (catalepsy, hypothermia, antinociception) in CB1 (+/+) mice, but these actions were essentially eliminated in CB1 (-/-) mice, indicating a CB1R mechanism of action. CB1R efficacy was inferred by comparing potencies between CB1 (+/+) and (+/-) mice [+/+ ED50 /+/- ED50], the latter of which has a 50% reduction of CB1Rs (i.e., decreased receptor reserve). Notably, CB1 (+/-) mice displayed profound rightward and downward shifts in the antinociception and hypothermia dose-response curves of low-efficacy compared with high-efficacy cannabinoids. In vitro efficacy, quantified using agonist-stimulated [35S]GTPγS binding in spinal cord tissue, significantly correlated with the relative efficacies of antinociception (r = 0.87) and hypothermia (r = 0.94) in CB1 (+/-) mice relative to CB1 (+/+) mice. Conversely, drug potencies for cataleptic effects did not differ between these genotypes and did not correlate with the in vitro efficacy measure. These results suggest that evaluation of antinociception and hypothermia in CB1 transgenic mice offers a useful in vivo approach to determine CB1R selectivity and efficacy of emerging SCs, which shows strong congruence with in vitro efficacy.

Inhibition of monoacylglycerol lipase reduces nicotine withdrawal.

BACKGROUND AND PURPOSE: Abrupt discontinuation of nicotine, the main psychoactive component in tobacco, induces a withdrawal syndrome in nicotine-dependent animals, consisting of somatic and affective signs, avoidance of which contributes to drug maintenance. While blockade of fatty acid amide hydrolase, the primary catabolic enzyme of the endocannabinoid arachidonoylethanolamine (anandamide), exacerbates withdrawal responses in nicotine-dependent mice, the role of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of a second endocannabinoid 2-arachidonylglycerol (2-AG), in nicotine withdrawal remains unexplored. EXPERIMENTAL APPROACH: To evaluate the role of MAGL enzyme inhibition in nicotine withdrawal, we initially performed a genetic correlation approach using the BXD recombinant inbred mouse panel. We then assessed nicotine withdrawal intensity in the mouse after treatment with the selective MAGL inhibitor, JZL184, and after genetic deletion of the enzyme. Lastly, we assessed the association between genotypes and smoking withdrawal phenotypes in two human data sets. KEY RESULTS: BXD mice displayed significant positive correlations between basal MAGL mRNA expression and nicotine withdrawal responses, consistent with the idea that increased 2-AG brain levels may attenuate withdrawal responses. Strikingly, the MAGL inhibitor, JZL184, dose-dependently reduced somatic and aversive withdrawal signs, which was blocked by rimonabant, indicating a CB1 receptor-dependent mechanism. MAGL-knockout mice also showed attenuated nicotine withdrawal. Lastly, genetic analyses in humans revealed associations of the MAGL gene with smoking withdrawal in humans. CONCLUSIONS AND IMPLICATIONS: Overall, our findings suggest that MAGL inhibition maybe a promising target for treatment of nicotine dependence.

Brain regional differences in CB1 receptor adaptation and regulation of transcription.

Cannabinoid CB1 receptors (CB1Rs) are expressed throughout the brain and mediate the central effects of cannabinoids, including Δ(9)-tetrahydrocannabinol (THC), the main psychoactive constituent of marijuana. Repeated THC administration produces tolerance to cannabinoid-mediated effects, although the magnitude of tolerance varies by effect. Consistent with this observation, CB1R desensitization and downregulation, as well as induction of immediate early genes (IEGs), vary by brain region. Zif268 and c-Fos are induced in the forebrain after acute THC administration. Phosphorylation of the cAMP response-element binding protein (CREB) is increased in a region-specific manner after THC administration. Results differ between acute versus repeated THC injection, and suggest that tolerance to IEG activation might develop in some regions. Repeated THC treatment produces CB1R desensitization and downregulation in the brain, although less adaption occurs in the striatum as compared to regions such as the hippocampus. Repeated THC treatment also induces expression of ΔFosB, a very stable isoform of FosB, in the striatum. Transgenic expression of ∆FosB in the striatum enhances the rewarding effects of several drugs, but its role in THC-mediated effects is not known. The inverse regional relationship between CB1R desensitization and ∆FosB induction suggests that these adaptations might inhibit each other, although this possibility has not been investigated. The differential regional expression of individual IEGs by acute or repeated THC administration suggests that regulation of target genes and effects on CB1R signaling will contribute to the behavioral effects of THC.

Statistical Parametric Mapping reveals ligand and region-specific activation of G-proteins by CB1 receptors and non-CB1 sites in the 3D reconstructed mouse brain.

CB(1) receptors mediate the CNS effects of Delta(9)-tetrahydrocannabinol and synthetic cannabinoids. Previous studies have investigated cannabinoid-mediated G-protein activity in a subset of brain regions thought to mediate the behavioral effects of cannabinoids, but a detailed regional comparison of the effects of multiple ligands has not been conducted. This study used a novel approach, Statistical Parametric Mapping (SPM), to analyze 3D reconstructed brain images derived from agonist-stimulated [(35)S]GTPgammaS autoradiography in a whole-brain unbiased manner. SPM analysis demonstrated regional differences in the relative efficacies of cannabinoid agonists methanandamide (M-AEA), CP55,940 (CP) and WIN55,212-2 (WIN) in CB(1)(+/+) mouse brains. To assess the potential contribution of novel cannabinoid binding sites, experiments were performed in CB(1)(-/-) mouse brains. SPM analysis revealed that the aminoalkylindole WIN, but not the bicyclic cannabinoid CP or the endocannabinoid analogue M-AEA, stimulated [(35)S]GTPgammaS binding in cortex, hippocampus, hypothalamus, amygdala, cerebellum and certain brainstem areas (dorsal tegmental complex and locus coeruleus). No differences between WIN-stimulated G-protein activity and basal activity were found in basal ganglia. Pharmacological experiments using the CB(1) antagonist SR141716A in CB(1)(+/+) mice showed that SR141716A blocked WIN-stimulated G-protein activity in all brain regions, suggesting that it binds to both CB(1) and putative non-CB(1) sites. These studies show ligand and region-specific cannabinoid-mediated G-protein activity at both CB(1) and non-CB(1) sites and demonstrate that SPM is a powerful approach for the analysis of reconstructed brain imaging data derived from agonist-stimulated [(35)S]GTPgammaS autoradiography.

Distinct patterns of DeltaFosB induction in brain by drugs of abuse.

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.

Region-dependent attenuation of mu opioid receptor-mediated G-protein activation in mouse CNS as a function of morphine tolerance.

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.

Status epilepticus causes a long-lasting redistribution of hippocampal cannabinoid type 1 receptor expression and function in the rat pilocarpine model of acquired epilepsy.

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.

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.

Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G-protein activation.

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.

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.

Inhibitory effects of SR141716A on G-protein activation in rat brain.

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), a cannabinoid CB(1) receptor antagonist, has inverse agonist effects in cannabinoid CB(1) receptor-expressing cell lines, brain and peripheral organs. These studies characterized SR141716A-inhibited G-protein activity by measuring [35S]GTPgammaS binding. Maximal inhibition of basal [35S]GTPgammaS binding in cerebellar membranes was 50%. The EC(50) value for inhibition of [35S]GTPgammaS binding was 4.4 microM, whereas the K(e) for inhibition of R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN 55,212-2)-stimulated [35S]GTPgammaS binding was 0.6 nM. [35S]GTPgammaS autoradiography was used to examine the regional specificity of SR141716A inhibition. SR141716A inhibited basal [35S]GTPgammaS binding in all regions examined, with inhibition ranging from approximately 20% in caudate-putamen to 40% in hippocampus. These studies demonstrate that SR141716A is a competitive antagonist at nanomolar concentrations, whereas it inhibits basal receptor-mediated G-protein activity at micromolar concentrations. These data suggest that the apparent inverse agonist effect is either not cannabinoid CB(1) receptor-specific or that SR141716A is binding to different sites on the cannabinoid CB(1) receptor to produce inverse agonist versus competitive antagonist effects.

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.

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.

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.

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.

Selective breeding of 5-HT(1A) receptor-mediated responses: application to emotion and receptor action.

Rat lines that were selectively bred for high (high DPAT-sensitive, HDS) or low (low DPAT-sensitive, LDS) hypothermic responses to the specific 5-HT(1A) receptor agonist, 8-hydroxy-di-n-propylaminotetralin (8-OH-DPAT), differ in receptor binding and certain behaviors related to anxiety and depression. After reviewing this literature, the present communication summarizes new experiments designed to clarify and extend the nature of the pharmacological and biochemical differences between the lines. A challenge with the 5-HT(2) receptor agonist, DOI, produced similar degrees of head shakes and skin crawls in the HDS and LDS rats, suggesting similar sensitivity of 5-HT(2A) and 5-HT(2C) receptors. In contrast, DOI-induced flat body posture (FBP), which has been linked to 5-HT(1A) receptor stimulation, was observed more readily in the HDS rats. The HDS and LDS rats exhibited similar degrees of increase in 8-OH-DPAT-stimulated [35S]GTPgammaS binding in several brain regions. This result suggests that the dramatic differences in hypothermia in HDS and LDS rats cannot be related to 5-HT(1A) receptor-mediated action on G proteins. Overall, these findings indicate that the selective breeding for 5-HT(1A)-mediated hypothermia has been fairly selective, and that differences in emotionally relevant behaviors between these two rat lines can strongly be associated with an unidentified component of the 5-HT(1A) receptor signaling pathway.

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.

Mu and kappa1 opioid-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding in cynomolgus monkey brain.

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.