Simultaneous absence of dopamine D1 and D2 receptor-mediated signaling is lethal in mice

Abstract

Dopamine (DA) controls a wide variety of physiological functions in the central nervous system as well as in the neuroendocrine and gastrointestinal systems. DA signaling is mediated by five cloned receptors named D1-D5. Knockout mouse models for the five receptors have been generated, and, albeit impaired for some important DA-mediated functions, they are viable and can reproduce. D1 and D2 receptors are the most abundant and widely expressed DA receptors. Cooperative/synergistic effects mediated by these receptors have been suggested, in particular, in the control of motor behaviors. To analyze the extent of such interrelationship, we have generated double D1/D2 receptor mutants. Interestingly, in contrast to single knockouts, we found that concurrent ablation of the D1 and D2 receptors is lethal during the second or third week after birth. This dramatic phenotype is likely to be related to altered feeding behavior and dysfunction of the gastrointestinal system, especially because major anatomical changes were not identified in the brain. Similarly, in the absence of functional D1, heterozygous D2 mutants (D1r(-/-);D2r(+/-)) showed severe growth retardation and did not survive their postweaning period. The analysis of motor behavior in D1r/D2r compound mutants showed that loss of D2-mediated functions reduces motor abilities, whereas the effect of D1r ablation on locomotion strongly depends on the experimental paradigms used. These studies highlight the interrelationship between D1 and D2 receptor-mediated control of motor activity, food intake, and gastrointestinal functions, which has been elusive in the single-gene ablation studies.

Fig. 1.

The contemporary knockout of D1 and D2 receptors is lethal. (A) Postnatal growth curves of WT (open squares) and DKO (filled squares) mice (n = 14). ^**, P < 0.01; ^***, P < 0.001 versus DKO mice (Student's t test). (B) Percentage of survival over the first three postnatal weeks of WT and DKO mice. No DKO survived past postnatal day 18 (n = 21). (C) Immunohistochemistry using anti-TH antibodies on brain sections from WT and DKO mice (as indicated). ST, striatum (scale bar = 600 μm); SN, substantia nigra (scale bar = 300 μm). No significant differences in the expression of this marker were detected. (D) Expression of striatal neuropeptides was evaluated by in situ hybridizations using mouse Enk, Dyn, and SP antisense probes. Enk expression is up-regulated, whereas Dyn and SP are down-regulated in DKO. (Scale bar = 750 μm.)

Fig. 2.

Poor intestinal development and hypothalamic dysfunctions in DKO. (A) Hematoxylin/eosin stainings of paraffin-embedded intestinal sections (duodenum) from WT (Left) and DKO (Right). (Scale bar, 400 μm.) (B) In situ hybridizations of brain sections from WT and DKO using probes specific for NPY, AGRP, and orexin. Animals were killed at the beginning of the dark phase when feeding behavior is nearly at its highest level. Experiments were repeated three times. WT values (black bars) were arbitrarily taken as 100%. ^*, P < 0.05; ^**, P < 0.01 versus WT (Student's t test). (Scale bar, 1.5 mm.)

Fig. 3.

The number of functional D2r alleles is critical for survival in a D1r^-/- background. (A) Growth curves of WT (n = 14), D1r^+/-;D2r^-/- (n = 14), and D1r^-/-;D2r^+/- (n = 11; fed with semiliquid diet, SLD) male mice and D1r^-/-;D2r^+/- (n = 8; fed with breeder diet, mice (BD). (B) Survival rates of D1r^-/-;D2r^+/+ (n = 34) and D1r^-/-;D2r^+/- male mice SLD (n = 29) or BD (n = 8). Survival rates for the other genotypes were between 93% and 100% (n = 25-47 per genotype). (C) D1r^-/-;D2r^+/- developed ulcer accompanied by intense hemorrhage under BD (Left) unless they were fed SLD (Right).

Fig. 4.

Microscopical evaluation of the histology of the duodenum of WT and compound mutants. (A) WT mice. (B) D1r^-/-;D2r^+/- mice fed with breeder diet. (C) D1r^-/-;D2r^+/- mice fed with semiliquid diet. Duodenal paraffin-embedded sections were stained with hematoxylin/eosin. A severe villous atrophy was observed in D1r^-/-;D2r^+/- mice fed with breeder diet. Lethal ulcerations appear to be prevented by semiliquid diet. (Scale bar, 200 μm.)

Fig. 5.

Analyses of motor activity of D1r/D2r compound mutants. (A) Motor activity of mutant mice (n = 24-27 per genotype) was assessed in the home cage. Beam breaks were collected during the dark phase of the light/dark cycle for 12 h. ^*, P < 0.05 versus WT (one-way ANOVA followed by Tukey's test). (B) Distance traveled (m) in the open field. Recordings were started during the first 60 min of exposure to the test. ^**, P < 0.01 versus WT (one-way ANOVA followed by Tukey's test). (C) Rearings were measured during the whole length of the open-field test. ^*, P < 0.05; ^**, P < 0.01 versus WT (one-way ANOVA followed by Tukey's test). (D) Ability to coordinate movements was assessed on the rotarod. ^**, P < 0.01 versus WT (one-way ANOVA followed by Tukey's test).