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To determine the palmitoylation sites in the human dopamine D(1) receptor, we expressed wild type and mutant receptors in which candidate cysteines in the carboxyl tail were substituted by alanines both individually (A347, A351) and together (AA). Our results showed that palmitoylation levels of A347 and A351 were reduced substantially and that AA had no detectable signal of palmitoylation. These data indicate that cysteines 347 and 351 were both palmitoylated and that they were the only sites of palmitoylation. We introduced a cAMP-dependent protein kinase site encompassing the position 351. We predicted that a functional cAMP-dependent protein kinase site would impair receptor-G protein coupling if it is not occluded by palmitoylation. Our results demonstrated that indeed, the introduction of the cAMP-dependent protein kinase site caused reduced potency of dopamine stimulation of adenylyl cyclase, and thus confirmed that when unoccluded, the cAMP-dependent protein kinase site introduced to position 351 of dopamine D(1) receptor could confer constitutive desensitization.

Dopamine D(1) receptors are expressed in the hippocampus and prefrontal cortex, suggesting a role in cognition. Dopamine D(1) receptor-deficient mice (D(1)-/-) were used to investigate the role of this receptor in spatial learning and memory. Using the Morris water maze, mice were trained to locate a hidden platform. Subsequently, the platform was removed from the maze and mice were scored for the percentage of time spent in the target quadrant and the number of crossings through the target position. D(1)-/- mice had significantly longer escape latencies compared to wild-type (D(1)+/+) and heterozygous (D(1)+/-) littermates and showed absence of spatial bias during the probe trials. In a visually cued task, D(1)-/- mice performed better than on the hidden platform trials, but maintained slightly higher escape latencies than D(1)+/+ and D(1)+/- mice. Naive D(1)-/- mice exposed only to the cued task eventually acquired identical escape latencies as the D(1)+/+ and D(1)+/- mice. Sensorimotor reflexes, locomotor activity, spontaneous alternation and contextual learning were not different among the groups. These results indicate that D(1)-/- mice have a deficit in spatial learning without visual or motor impairment, suggesting that dopamine D(1) receptors are involved in at least one form of the cognitive processes.

The DBA/2J and C57BL/6J (herein referred to as DBA and C57) inbred mouse strains exhibit low and high predispositions for voluntary ethanol consumption, respectively, but the neurobiological basis underlying this differential drug vulnerability remains poorly understood. Comparison of endogenous brain proenkephalin gene expression showed the C57 mouse, compared to the DBA mouse, had lower preproenkephalin mRNA abundance, proenkephalin concentration and processed [Met5]enkephalin-immunoreactive peptide levels in the mid brain. No strain differences in enkephalin gene expression was observed in the striatum, hypothalamus, or medulla pons. Neurochemical analysis of C57 mice, following high voluntary ethanol consumption (approximately 17 g/kg/day), revealed markedly higher enkephalin gene expression in the striatum and mid brain compared to ethanol-naive animals. These findings suggested that mesolimbic enkephalin is augmented following ethanol consumption, and that endogenous low enkephalin biosynthesis may be associated with an increased vulnerability for ethanol abuse. However, the neurobiological basis of this behaviour may not be quite this simple. C57 mice pretreated with the dopamine receptor agonist, bromocriptine, had reduced striatum and mid brain preproenkephalin mRNA levels, and showed a 41% lower voluntary ethanol consumption compared to controls. We conclude that functional connectivity exists between enkephalin and dopamine systems, and although low mesolimbic enkephalin may predispose to high ethanol preference, dopamine is a more important determinant than enkephalin in the hierarchy of neurotransmitter pathways that mediate the increased vulnerability for ethanol consumption in the C57 mouse.

The propensity for high ethanol preference and high ethanol consumption (herein referred to as ethanol abuse) may be a consequence of a congenital deficit in central dopaminergic activity. This hypothesis was examined in the ethanol-avoiding DBA/2J (DBA) and ethanol-preferring C57BL/6J (C57) inbred mouse strains. Endogenous dopamine D1 and D2 receptor functions differed between strains in the nigrostriatal/mesolimbic dopamine system. At the level of the forebrain, the C57 mouse exhibited higher dopamine D1 and D2 receptor mRNA abundance and elevated dopamine D1 and D2 receptor densities in the striatum compared to DBA mouse. A likely explanation for these observations might be that higher dopamine receptor gene expression could be a consequence of low synaptic dopamine activity. Accordingly, we found higher striatal dopamine-sensitive adenylyl cyclase activity in the C57 mouse. The C57 mouse exhibited an enhanced dopamine D1-D2 receptor link as suggested by an enhanced up-regulation of striatal dopamine D2 receptor mRNA following dopamine D1 receptor blockade with SCH-23390 compared to DBA mouse. At the level of the mesencephalon and hind brain, the C57 mouse had lower dopamine D2 receptor mRNA in the medulla pons, and correspondingly lower midbrain and medulla pons dopamine D2 receptor densities. Adenylyl cyclase activities in these regions were similar to the DBA mouse suggesting that the coupling of these dopamine D2 receptors could be a factor regulating their function. Strain differences in dopamine D2 receptor function were also observed in the diencephalic dopamine system. The C57 mouse exhibited lower dopamine D2 receptor density in the hippocampus and lower dopamine D2 receptor mRNA abundance and lower adenylyl cyclase activity in the hypothalamus. Changes in brain dopamine receptor gene expression following ethanol intake inferred an increase in the activities of central dopamine pathways in both the DBA and C57 mouse supporting an association between dopamine receptor function and ethanol drinking. These lines of evidence provide a basis for the hypothesis that a genetically determined brain dopaminergic deficit mediated by dopamine D1-D2 receptor mechanisms may be involved in at least a part of the risk for ethanol abuse in the C57 inbred mouse strain.

This report experimentally examined whether the genetically determined low nigrostriatal/mesolimbic dopaminergic activity in the C57BL/6J (herein referred to as C57) inbred mouse mediated the congenital high risk for ethanol abuse (ethanol consumption and ethanol preference) in this model. C57 mice pretreated with dopamine D1 receptor agonist ((+)-SKF-38393) or dopamine D2 receptor agonist (bromocriptine) to augment synaptic dopamine availability exhibited marked 76% and 38% reductions in voluntary ethanol intake in comparison to untreated controls. Dopamine receptor agonist administration resulted in changes in dopamine D1 and D2 receptor mRNA in the cell bodies and dopamine D1 and D2 receptor densities principally in the afferent targets of nigrostriatal/mesolimbic dopamine neurons. Dopamine receptor agonists promoted a decrease of striatal dopamine D1 and D2 receptor densities and corresponding down-regulation of olfactory tubercle dopamine D1 and D2 receptor mRNA abundance. Dopamine receptor agonist-induced increases in forebrain dopaminergic activity was compensated with increased dopamine D2 receptor density and correspondingly higher dopamine D2 receptor mRNA content in the brain stem. When bromocriptine was administered to ethanol-sensitized mice, it was ineffective in reducing voluntary ethanol abuse. In these mice, treatment with the dopamine D2 receptor antagonist haloperidol led to a 28% reduction in the absolute amount of ethanol consumed, but not in voluntary ethanol preference. These data indicated that nigrostriatal/mesolimbic dopamine D1-D2 receptor mechanism(s) mediating the potential for becoming high ethanol drinking on exposure to ethanol are distinct from factors mediating voluntary ethanol drinking after sensitization to ethanol. These data constitute direct evidence supporting a dopamine hypothesis for ethanol abuse in the genetically ethanol-preferring C57 mouse.

The regulation and post-translational modifications of the human dopamine D1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D1 receptor or a DNA encoding a c-myc epitope tagged dopamine D1 receptor (c-myc-dopamine D1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D1 and c-myc-dopamine D1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D1 receptor, the photoaffinity label [125I](3-methyl-2-[4'-azidophenyl]-2,3,5-tetrahydro-2H-3-benzazepine) ([125I]MAB) bound specifically upon photolysis. A major broad band of approximately 48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D1 receptor was isolated by immunoprecipitation from whole cells and was shown to be post-translationally modified by phosphorylation and palmitoylation. Exposure of cells expressing c-myc-dopamine D1 receptor to dopamine for 15 min resulted in a reduction in the maximal dopamine stimulated adenylyl cyclase activity, which was accompanied by an increased phosphorylation of the receptor and a rapid redistribution of surface c-myc-dopamine D1 receptor as detected by in situ immunofluorescence. Dopamine exposure also resulted in an increased level of incorporation of [3H]palmitic acid into the receptor. Thus, we provide the first evidence that the human dopamine D1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.

[Met5]Enkephalin concentrations in rat pituitary were examined following the administration of the opiate antagonist naloxone 5 mg/kg per day as single daily s.c. injections for 9 days. Compared to vehicle administration, there was a significant increase of [Met5]enkephalin concentrations in both pituitary lobes, ranging from 50-90% above control values. Similar treatment with the opioid agonist [D-Ala2,MePhe4,Met(o)5ol]enkephalin in doses up to 500 micrograms/kg per day did not result in any alteration of [Met5]enkephalin levels. These data suggest that opioid receptor mechanisms play a role in maintaining [Met5]enkephalin concentrations in both neurointermediate and anterior pituitary.