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The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

We studied the functional activation of different polymorphic variants of the human dopamine D(4) receptors by the three major central monoamines, dopamine, noradrenaline and serotonin. Dopamine D(4) receptors carrying two (D4.2), four (D4.4) or seven (D4.7) repeats within the third intracellular domain were co-expressed with G protein-regulated inwardly rectifying potassium channels (GIRK1) in frog oocytes. All the dopamine D(4) receptor variants coupled to oocyte G(i/o) proteins and modulated co-expressed GIRK1 channels. Monoamine-induced responses were detected as increases in voltage-clamp recorded GIRK1 currents. Dopamine, noradrenaline as well as serotonin stimulated dopamine D(4) receptors. Dose-response analysis showed that dopamine and noradrenaline are full agonists whereas serotonin acted as partial agonist. Dopamine was 5-fold more potent on D4.2 and D4.7 (EC(50)=1 nM) than on D4.4 (EC(50)=5 nM) suggesting that the actions of dopamine and therapeutic drugs on dopamine D(4) receptors might vary among individuals depending on their repertoire of expressed alleles. In contrast, noradrenaline and serotonin did not discriminate among dopamine D(4) receptor variants (EC(50 NA)=50 nM, EC(50 5-HT)=1.5 microM). All monoamine effects were blocked by the specific dopaminergic D(4) antagonist (S)-(-)-4-[4-[2-(Isochroman-1-yl)ethyl]piperazin-1-yl]benzenesulfonamide (PNU101387). Sequence analyses of dopamine D(4) receptors and related monoamine receptors revealed that dopamine D(4) receptors have most aminoacidic residues necessary for binding of dopamine, noradrenaline and serotonin. Our data indicate that dopamine D(4) receptors can be pharmacologically stimulated by any the three major central monoamines.

Dopamine receptor type 2 (D2R) knockout mice (KO) have chronic hyperprolactinemia, pituitary hyperplasia, and a moderate decrease in MSH content. They are also growth retarded evidencing an alteration in the GH-IGF-I axis. In D2R KO, lactotropes do not show dense secretory granules but degranulated cells and fewer somatotropes, gonadotropes and thyrotropes. Prolactin levels are always higher in female than in male knockouts, and in accordance, pituitary hyperplasia is observed at 8 months only in females. After 16 months of age, highly vascularized adenomas develop, especially in females. Prominent vascular channels in the hyperplastic and adenomatous pituitaries, as well as extravasated red blood cells not contained in capillaries is also a common finding. Prolactin is not the factor that enhances the hyperplastic phenotype in females while estrogen is a permissive factor. VEGF-A expression is increased in pituitaries from D2R KO. VEGF-A is expressed in follicle stellate cells. Because D2R receptors are found in lactotropes and not in follicle stellate cells, it may be inferred that a paracrine-derived factor from lactotropes is acting on follicle stellate cells to increase VEGF-A expression. VEGF-A does not induce pituitary cell proliferation, even though it enhances prolactin secretion. But it may act on adjacent endothelial cells and participate in the angiogenic process that increases the availability of different growth factors and mitogens. The D2R knockout mouse represents a unique animal model to study dopamine-resistant prolactinomas, and VEGF-A may be an alternative therapeutic target in this pathology.

Using a novel system, the role of D2-like dopamine receptors in distinct topographies of orofacial movement was assessed in mutant mice with congenic D2 vs. D3 receptor knockout, and compared with findings in D1A mutants. Under spontaneous conditions, D2 mutants evidenced increased vertical jaw movements and unaltered horizontal jaw movements, with reductions in tongue protrusions and incisor chattering; in D3 mutants, only incisor chattering was reduced. Given previous evidence that D1A mutants show reduced horizontal but not vertical jaw movements, this indicates that apparent oppositional D1-like:D2-like interactions in the regulation of composited jaw movements may in fact reflect the independent actions of D2 receptors to inhibit vertical jaw movements and of D1A receptors to facilitate horizontal jaw movements. Effects of the D2-like agonist RU 24213 to exert greater reduction in horizontal than in vertical jaw movements were not altered prominently in either D2 or D3 mutants. The D1-like agonists A 68930 and SK&F 83959 induced vertical jaw movements, tongue protrusions, and incisor chattering; induction of tongue protrusions by A 68930 was reduced in D2 mutants. D2 receptors exert topographically specific regulation of orofacial movements in a manner distinct from their D1A counterparts, while D3 receptors exert only minor regulation of such movements., (Copyright 2003 Wiley-Liss, Inc.)

Rationale: Previous studies have found a role for dopamine D(2)-like receptors in many of the behavioral effects of cocaine, including its stimulation of locomotor activity and interoceptive discriminative-stimulus effects. However, given the lack of selectivity of most of the available pharmacological tools among D(2), D(3) and D(4) dopamine receptors, the roles of these specific receptors remain unclear., Objectives: The roles of specific dopamine D(4) receptors in the behavioral effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects were investigated using dopamine D(4) receptor knockout (DA D(4)R KO) and wild-type (WT) mice., Methods: The mice were trained in daily sessions to discriminate IP injections of saline from cocaine (10 mg/kg). Responses on one of two response keys intermittently produced a food pellet; one response was reinforced in sessions following cocaine injection (10 mg/kg), and the other response was reinforced in sessions following saline injection. Each 20th response produced a food pellet (fixed-ratio, or FR20 schedule of reinforcement). The dose-effects of cocaine and its interaction with the D(2)-like antagonist, raclopride, were assessed. Horizontal locomotor activity was also assessed in each genotype., Results: As previously shown), cocaine was a more potent stimulant of locomotor activity in the DA D(4)R KO mice compared to WT littermate mice. In addition, cocaine was more potent in producing discriminative-stimulus effects in DA D(4)R KO mice (ED(50) value=0.50 mg/kg) compared to their WT littermates (ED(50) value=2.6 mg/kg). Raclopride shifted the cocaine dose-effect curve in both DA D(4)R KO and WT mice, though the shift was greater for the DA D(4)R KO mice., Conclusions: The present results on the stimulation of activity and interoceptive/subjective effects of cocaine are consistent with the previously reported disregulation of dopamine synthesis in DA D(4)R KO mice, and further suggest a role of the DA D(4)R in vulnerability to stimulant abuse.

Feeding behavior can be divided into appetitive and consummatory phases, differing in neural substrates and effects of deprivation. Opioids play an important role in the appetitive aspects of feeding, but they also have acute stimulatory effects on food consumption. Because the opioid peptide beta-endorphin is co-synthesized and released with melanocortins from proopiomelanocortin (POMC) neuronal terminals, we examined the physiological role of beta-endorphin in feeding and energy homeostasis using a strain of mutant mice with a selective deficiency of beta-endorphin. Male beta-endorphin-deficient mice unexpectedly became obese with ad libitum access to rodent chow. Total body weight increased by 15% with a 50-100% increase in the mass of white fat. The mice were hyperphagic with a normal metabolic rate. Despite the absence of endogenous beta-endorphin, the mutant mice did not differ from wild-type mice in their acute feeding responses to beta-endorphin or neuropeptide Y administered intracerebroventricularly or naloxone administered intraperitoneally. Additional mice were studied using an operant behavioral paradigm to examine their acquisition of food reinforcers under increasing work demands. Food-deprived, beta-endorphin-deficient male mice emitted the same number of lever presses under a progressive ratio schedule compared to wild-type mice. However, the mutant mice worked significantly less than did the wild-type mice for food reinforcers under nondeprived conditions. Controls for nonspecific effects on acquisition of conditioned learning, activity, satiety, and resistance to extinction revealed no genotype differences, supporting our interpretation that beta-endorphin selectively affects a motivational component of reward behavior under nondeprived conditions. Therefore, we propose that beta-endorphin may function in at least two primary modes to modulate feeding. In the appetitive phase, beta-endorphin release increases the incentive value of food as a primary reinforcer. In contrast, it appears that endogenous beta-endorphin may inhibit food consumption in parallel with melanocortins and that the orexigenic properties previously ascribed to it may actually be due to other classes of endogenous opioid peptides.

Proopiomelanocortin (POMC) neurons in the hypothalamus are direct targets of the adipostatic hormone leptin and contribute to energy homeostasis by integrating peripheral and central information. The melanocortin and beta-endorphin neuropeptides are processed from POMC and putatively coreleased at axon terminals. Melanocortins have been shown by a combination of pharmacological and genetic methods to have inhibitory effects on appetite and body weight. In contrast, pharmacological studies have generally indicated that opioids stimulate food intake. Here we report that male mice engineered to selectively lack beta-endorphin, but that retained normal melanocortin signaling, were hyperphagic and obese. Furthermore, beta-endorphin mutant and wild-type mice had identical orexigenic responses to exogenous opioids and identical anorectic responses to the nonselective opioid antagonist naloxone, implicating an alternative endogenous opioid tone to beta-endorphin that physiologically stimulates feeding. These genetic data indicate that beta-endorphin is required for normal regulation of feeding, but, in contrast to earlier reports suggesting opposing actions of beta-endorphin and melanocortins on appetite, our results suggest a more complementary interaction between the endogenously released POMC-derived peptides in the regulation of energy homeostasis.

Unlabelled: The use of mice with targeted gene deletions (knockouts [KOs]) provides an important tool to investigate the mechanisms underlying behavior, neuronal development, and the sequella of neuropsychiatric diseases. MRI has been used to image brain structural changes in KO mice but, to our knowledge, the feasibility of using PET to investigate brain neurochemistry in KO mice has not been demonstrated., Methods: We have evaluated the sensitivity of the microPET to image dopamine D2 receptor (DRD2) KO mice (D2-/-). PET measurements were performed in wild-type (D2+/+) mice and KO (D2-/-) mice using a microPET scanner. Briefly, each animal was anesthetized and injected intravenously with (11)C-raclopride, a DRD2-specific ligand, and dynamic PET scanning was performed for 60 min., Results: The (11)C-raclopride images of the KO mice showed significantly lower binding in the striatum (ST) than those of the wild-type (WT) mice, which was confirmed by the time-activity curves that revealed equivalent binding in the ST and cerebellum (CB) in KO mice, whereas the WT mice had significantly higher binding in the ST than in the CB. The ST/CB ratio was significantly higher in WT mice than in KO mice (ST/CB = 1.33 +/- 0.13 and 1.05 +/- 0.03, respectively; P < 0.002; n = 10). The microPET images were compared qualitatively with conventional autoradiography images., Conclusion: These data support the use of microPET as an effective in vivo imaging tool for studying noninvasively KO mice. These same tools can be extended to investigate other genetically engineered murine models of disease. Future studies will seek to use microPET to investigate the relationships between genes, neuronal activity, and behavior.

We used beta-endorphin-deficient mice as a novel approach to confirm the physiological role that opioid peptides play in the development or regulation of the immune system. We found that mice lacking beta-endorphin possessed an enhanced immune response, measured in terms of splenocyte proliferation and interleukin (IL)-2 mRNA levels, in vitro production of the splenic macrophage inflammatory cytokines IL-6 and Tumor Necrosis Factor (TNF)-alpha and plasma IL-6 following lipopolysaccharide (LPS) administration. beta-Endorphin-deficient mice had attenuated increases of plasma ACTH and corticosterone levels in response to LPS. These results are consistent with a postulated inhibitory role of endogenous beta-endorphin on the immune system at multiple levels.

Objective: To assess whether D(2) receptors participate in the regulation of genetic hypertension., Methods: Arterial pressure was measured in mice mutant for the D(2) dopamine receptors, and the interactions between D(2) dopamine receptors and other vasopressor systems were also studied., Results: Both systolic blood pressure (BP) in D(2) mutant (D(2)-/-) and hybrid (D(2)+/-) mice (128 +/- 2 mm Hg, 129 +/- 4 mm Hg) and diastolic BP (107 +/- 2 mm Hg, 108 +/- 3 mm Hg) were higher than in wild-type (D(2)+/+) mice (104 +/- 2 mm Hg, 77 +/- 1 mm Hg). The BP after alpha-adrenergic blockade decreased to a greater extent in D(2)-/- than in D(2)+/+ mice. Epinephrine excretion was greater in D(2)-/- than in D(2)+/+ mice and adrenalectomy decreased blood pressure to a greater extent in D(2)-/- than in D(2)+/+ mice. The non specific endothelin B (ETB) blocker (BQ788) decreased BP in D(2)-/- but had not in D(2)+/+ mice. The ETB1 blocker RES 701 - 1 increased BP in D(2)-/- but not D(2)+/+ mice. ET-1 and the ETB agonist, sarafatoxin S6c increased BP to a greater extent in D(2)+/+ than in D(2)-/- mice. Circulating ET-like immunoreactive peptides were similar in D(2)-/- and D(2)+/+ mice but ETB receptor expression was greater in D(2)-/- than in D(2)+/+ mice. In contrast, blockade of ETA and V(1) vasopressin receptors had no effect on BP in either D(2)-/- and D(2)+/+ mice. The hypotensive effect of the AT(1) antagonist, losartan, was also similar in D(2)-/- and D(2)+/+ mice. The greater basal sodium excretion and lower renal Na(+)/K(+)ATPase activity in D(2)-/- than in D(2)+/+ mice indicate that sodium retention was not the cause of hypertension in these animals., Conclusion: Hypertension in the D(2) mutant mice may be caused by absent inhibitory effect of D(2) receptors on sympathetic outflow coupled with increased ETB2 activity.