Adenosine and dopamine signaling exert opposite effects in the basal ganglia, a brain region involved in sensory-motor integration. Thus, adenosine agonists induce motor depression and adenosine antagonists, such as caffeine, produce motor activation (1). These opposite effects result from specific antagonistic interactions between subtypes of adenosine and dopamine receptors in the striatum, the main input structure of the basal ganglia. In fact, striatal dopamine receptors and, to some extent, adenosine receptors are segregated in the two main populations of ␥-aminobutyric acid (GABA) efferent neurons. EXPERIMENTAL PROCEDURESCell Cultures-Maintenance of SH-SY5Y cells (parental and D 2 Rtransfected cells) as well as the pharmacological characterization and maintenance of D 2 R-and D 1 R-transfected mouse fibroblast Ltk Ϫ cells are described in detail elsewhere (7-9). For primary cultures, striata were removed from 16-day-old Sprague-Dawley rat embryos (B&K Universal) in Ca 2ϩ /Mg 2ϩ -free PBS supplemented with 20 units/ml penicillin and 20 g/ml streptomycin (Invitrogen). The tissue fragments were pooled and mechanically dissociated in SFM Neurobasal serum-free medium (Invitrogen), supplemented with B27 (Invitrogen), glutamine (2 mM; Invitrogen), penicillin/streptomycin (20 units/ml/20 g/ml; Invitrogen), and ␤-mercaptoethanol (25 M) (Invitrogen). Cells were collected by centrifugation at 100 ϫ g for 5 min and resuspended in fresh medium. The resulting single-cell suspension was seeded on 24-well plates coated with gelatin (Sigma) and poly-L-lysine (Sigma), and cells were grown at 37°C in saturation humidity with 5% CO 2 .Immunolabeling Experiments-Neuroblastoma cells were grown on glass coverslips coated with poly-L-lysine (Sigma) and exposed to vari-* This work was

Alcoholism is a chronic relapsing disorder with substantial heritability. Uncovering gene-environment interactions underlying this disease process can aid identification of novel treatment targets. Here, we found a lowered threshold for stress-induced reinstatement of alcohol seeking in Marchigian-Sardinian Preferring (msP) rats genetically selected for high alcohol preference. In situ hybridization for a panel of 20 stress-related genes in 16 brain regions was used to screen for differential gene expression that may underlie this behavioral phenotype. An innate up-regulation of the Crhr1 transcript, encoding the corticotropin-releasing hormone receptor 1 (CRH-R1), was found in several limbic brain areas of msP rats genetically selected for high alcohol preference, was associated with genetic polymorphism of the Crhr1 promoter, and was accompanied by increased CRH-R1 density. A selective CRH-R1 antagonist (antalarmin, 10 -20 mg͞kg) was devoid of effects on operant alcohol self-administration in unselected Wistar rats but significantly suppressed this behavior in the msP line. Stressinduced reinstatement of alcohol seeking was not significantly affected by antalarmin in Wistar rats but was fully blocked in msP animals. These data demonstrate that Crhr1 genotype and expression interact with environmental stress to reinstate alcohol-seeking behavior.lcohol use is the number three modifiable cause of death in the United States (1). Alcohol dependence, hereafter called alcoholism, is a complex behavioral disorder in which substantial heritable susceptibility factors interact with the environment to produce and maintain the disease state (2). Alcoholism is clinically characterized by a chronic relapsing course similar to other common medical conditions (3). Relapse, i.e., return to alcohol seeking and uncontrolled drinking after varying intervals of sobriety, is a key phenomenon in this process, making relapse prevention a primary therapeutic objective.Gene-environment interactions are commonly implicated in alcoholism and propensity to relapse, but their exact nature is presently unknown. A behavioral analysis has long pointed to three broad categories of environmental stimuli with an ability to trigger relapse in susceptible individuals (4): consumption of small, ''priming'' doses of alcohol, presentation of conditioned cues associated with prior availability of alcohol, and stress. It is unclear whether, in alcohol-dependent individuals, these stimuli trigger relapse by interacting with preexisting genetic susceptibility factors, acquired CNS neuroadaptations secondary to a prolonged history of alcohol use, or both.Models in experimental animals offer tools in the search for novel alcoholism treatments (5, 6) and may be helpful in addressing this question. Genetic selection for high alcohol preference in rats has resulted in several lines with pharmacologically relevant levels of voluntary intake of alcohol, as well as other alcohol-related phenotypes (7,8), and an improved understanding of mechanisms mediating rel...

The involvement of adenosine A 1 and A 2A receptors in the motor effects of caffeine is still a matter of debate. In the present study, counteraction of the motor-depressant effects of the selective A 1 receptor agonist CPA and the A 2A receptor agonist CGS 21680 by caffeine, the selective A 1 receptor antagonist CPT, and the A 2A receptor antagonist MSX-3 was compared. CPT and MSX-3 produced motor activation at the same doses that selectively counteracted motor depression induced by CPA and CGS 21680, respectively. Caffeine also counteracted motor depression induced by CPA and CGS 21680 at doses that produced motor activation. However, caffeine was less effective than CPT at counteracting CPA and even less effective than MSX-3 at counteracting CGS 21680. On the other hand, when administered alone in habituated animals, caffeine produced stronger motor activation than CPT or MSX-3. An additive effect on motor activation was obtained when CPT and MSX-3 were coadministered. Altogether, these results suggest that the motoractivating effects of acutely administered caffeine in rats involve the central blockade of both A 1 and A 2A receptors. Chronic exposure to caffeine in the drinking water (1.0 mg/ml) resulted in tolerance to the motor effects of an acute administration of caffeine, lack of tolerance to amphetamine, apparent tolerance to MSX-3 (shift to the left of its 'bell-shaped' dose-response curve), and true crosstolerance to CPT. The present results suggest that development of tolerance to the effects of A 1 receptor blockade might be mostly responsible for the tolerance to the motor-activating effects of caffeine and that the residual motor-activating effects of caffeine in tolerant individuals might be mostly because of A 2A receptor blockade.

Recently evidence has been presented that adenosine A2A and dopamine D2 receptors form functional heteromeric receptor complexes as demonstrated in human neuroblastoma cells and mouse fibroblast Ltk- cells. These A2A/D2 heteromeric receptor complexes undergo coaggregation, cointernalization, and codesensitization on D2 or A2A receptor agonist treatments and especially after combined agonist treatment. It is hypothesized that the A2A/D2 receptor heteromer represents the molecular basis for the antagonistic A2A/D2 receptor interactions demonstrated at the biochemical and behavioral levels. Functional heteromeric complexes between A2A and metabotropic glutamate 5 receptors (mGluR5) have also recently been demonstrated in HEK-293 cells and rat striatal membrane preparations. The A2A/mGluR5 receptor heteromer may account for the synergism found after combined agonist treatments demonstrated in different in vitro and in vivo models. D2, A2A, and mGluR5 receptors are found together in the dendritic spines of the striatopallidal GABA neurons. Therefore, possible D2/A2A/mGluR5 multimeric receptor complexes and the receptor interactions within them may have a major role in controlling the dorsal and ventral striatopallidal GABA neurons involved in Parkinson's disease and in schizophrenia and drug addiction, respectively.

The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A -D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the I3 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of I3 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson's disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.

Background-Alcoholism is a complex behavioral disorder in which interactions between stressful life events and heritable susceptibility factors contribute to the initiation and progression of disease. Neural substrates of these interactions remain largely unknown. Here, we examined the role of the nociceptin/orphanin FQ (N/OFQ) system, using an animal model in which genetic selection for high alcohol preference has led to co-segregation of elevated behavioral sensitivity to stress (msP rats).

In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor-receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor-receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor-receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor-receptor interactions namely the presence of regulated cooperativity via receptor-receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor-receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor-receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor-receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson's disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor-receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.

Wolfram syndrome (WS) is a rare autosomal-recessive disorder that is caused by mutations in the WFS1 gene and is characterized by juvenile-onset diabetes, optic atrophy, hearing loss and a number of other complications. Here, we describe the creation and phenotype of Wfs1 mutant rats, in which exon 5 of the Wfs1 gene is deleted, resulting in a loss of 27 amino acids from the WFS1 protein sequence. These Wfs1-ex5-KO232 rats show progressive glucose intolerance, which culminates in the development of diabetes mellitus, glycosuria, hyperglycaemia and severe body weight loss by 12 months of age. Beta cell mass is reduced in older mutant rats, which is accompanied by decreased glucose-stimulated insulin secretion from 3 months of age. Medullary volume is decreased in older Wfs1-ex5-KO232 rats, with the largest decreases at the level of the inferior olive. Finally, older Wfs1-ex5-KO232 rats show retinal gliosis and optic nerve atrophy at 15 months of age. Electron microscopy revealed axonal degeneration and disorganization of the myelin in the optic nerves of older Wfs1-ex5-KO232 rats. The phenotype of Wfs1-ex5-KO232 rats indicates that they have the core symptoms of WS. Therefore, we present a novel rat model of WS.