Central dopamine D2 receptors regulate growth-hormone-dependent body growth and pheromone signaling to conspecific males.

Competition between adult males for limited resources such as food and receptive females is shaped by the male pattern of pituitary growth hormone (GH) secretion that determines body size and the production of urinary pheromones involved in male-to-male aggression. In the brain, dopamine (DA) provides incentive salience to stimuli that predict the availability of food and sexual partners. Although the importance of the GH axis and central DA neurotransmission in social dominance and fitness is clearly appreciated, the two systems have always been studied unconnectedly. Here we conducted a cell-specific genetic dissection study in conditional mutant mice that selectively lack DA D2 receptors (D2R) from pituitary lactotropes (lacDrd2KO) or neurons (neuroDrd2KO). Whereas lacDrd2KO mice developed a normal GH axis, neuroDrd2KO mice displayed fewer somatotropes; reduced hypothalamic Ghrh expression, pituitary GH content, and serum IGF-I levels; and exhibited reduced body size and weight. As a consequence of a GH axis deficit, neuroDrd2KO adult males excreted low levels of major urinary proteins and their urine failed to promote aggression and territorial behavior in control male challengers, in contrast to the urine taken from control adult males. These findings reveal that central D2Rs mediate a neuroendocrine-exocrine cascade that controls the maturation of the GH axis and downstream signals that are critical for fitness, social dominance, and competition between adult males.

Cocaine supersensitivity and enhanced motivation for reward in mice lacking dopamine D2 autoreceptors.

Dopamine (DA) D2 receptors expressed in DA neurons (D2 autoreceptors) exert a negative feedback regulation that reduces DA neuron firing, DA synthesis and DA release. As D2 receptors are mostly expressed in postsynaptic neurons, pharmacological and genetic approaches have been unable to definitively address the in vivo contribution of D2 autoreceptors to DA-mediated behaviors. We found that midbrain DA neurons from mice deficient in D2 autoreceptors (Drd2(loxP/loxP); Dat(+/IRES-cre), referred to as autoDrd2KO mice) lacked DA-mediated somatodendritic synaptic responses and inhibition of DA release. AutoDrd2KO mice displayed elevated DA synthesis and release, hyperlocomotion and supersensitivity to the psychomotor effects of cocaine. The mice also exhibited increased place preference for cocaine and enhanced motivation for food reward. Our results highlight the importance of D2 autoreceptors in the regulation of DA neurotransmission and demonstrate that D2 autoreceptors are important for normal motor function, food-seeking behavior, and sensitivity to the locomotor and rewarding properties of cocaine.

Fibroblast growth factor-2 in hyperplastic pituitaries of D2R knockout female mice.

Dopamine D2 receptor (D2R) knockout (KO) female mice develop chronic hyperprolactinemia and pituitary hyperplasia. Our objective was to study the expression of the mitogen fibroblast growth factor (FGF2) and its receptor, FGFR1, comparatively in pituitaries from KO and wild-type (WT) female mice. We also evaluated FGF2 subcellular localization and FGF2 effects on pituitary function. FGF2-induced prolactin release showed a similar response pattern in both genotypes, even though basal and FGF2-stimulated release was higher in KO. FGF2 stimulated pituitary cellular proliferation (MTS assay and [(3)H]thymidine incorporation), with no differences between genotypes. FGF2 concentration (measured by ELISA) in whole pituitaries or cultured cells was lower in KO (P < 0.00001 and 0.00014). Immunofluorescence histochemistry showed less FGF2 in pituitaries from KO females and revealed a distinct FGF2 localization pattern between genotypes, being predominantly nuclear in KO and cytosolic in WT pituitaries. Finally, FGF2 could not be detected in the conditioned media from pituitary cultures of both genotypes. FGFR1 levels (Western blot and immunohistochemistry) were higher in pituitaries of KO. Basal concentration of phosphorylated ERKs was lower in KO cells (P = 0.018). However, when stimulated with FGF2, a significantly higher increment of ERK phosphorylation was evidenced in KO cells (P < or = 0.02). We conclude that disruption of the D2R caused an overall decrease in pituitary FGF2 levels, with an increased distribution in the nucleus, and increased FGFR1 levels. These results are important in the search for reliable prognostic indicators for patients with pituitary dopamine-resistant prolactinomas, which will make tumor-specific therapy possible.

Dopaminergic D2 receptor knockout mouse: an animal model of prolactinoma.

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.

Increased pituitary vascular endothelial growth factor-a in dopaminergic D2 receptor knockout female mice.

Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [(3)H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R(-/-) cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.

Dopamine D4 receptor-deficient mice display cortical hyperexcitability.

The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.

Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine.

The human dopamine D4 receptor (D4R) has received considerable attention because of its high affinity for the atypical antipsychotic clozapine and the unusually polymorphic nature of its gene. To clarify the in vivo role of the D4R, we produced and analyzed mutant mice (D4R-/-) lacking this protein. Although less active in open field tests, D4R-/- mice outperformed wild-type mice on the rotarod and displayed locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Biochemical analyses revealed that dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from D4R-/- mice. Based on these findings, we propose that the D4R modulates normal, coordinated and drug-stimulated motor behaviors as well as the activity of nigrostriatal dopamine neurons.