Marys Medicine

5834 • The Journal of Neuroscience, March 27, 2013 • 33(13):5834 –5842
Central Dopamine D2 Receptors Regulate Growth-Hormone-
Dependent Body Growth and Pheromone Signaling to
Conspecific Males

Daniela Noaín,1* M. Ine´s Pe´rez-Milla´n,2* Estefanía P. Bello,1 Guillermina M. Luque,2 Rodrigo Casas Cordero,1
Diego M. Gelman,1 Marcela Peper,1 Isabel García Tornadu,2 Malcolm J. Low,3 Damasia Becu´-Villalobos,2
and Marcelo Rubinstein1,4
1Instituto de Investigaciones en Ingeniería Gene´tica y Biología Molecular, and 2Instituto de Biología y Medicina Experimental, Consejo Nacional de
Investigaciones Científicas y Te´cnicas, C1428ADN Buenos Aires, Argentina, 3Department of Molecular and Integrative Physiology, University of Michigan
Medical School, Ann Arbor, Michigan 48105, and 4Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires,
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 aggres-
sion. 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.

appreciated in mice lacking D2Rs (Drd2 ⫺/⫺) that display hyper- Central D2 receptors (D2Rs) participate in important brain func- prolactinemia and develop pituitary hyperplasia tions, including the control of locomotor activity and reward- Analysis of Drd2 ⫺/⫺ mice also revealed the seeking behavior. D2Rs are also found in pituitary lactotropes, unexpected importance of D2Rs in the regulation of the growth where they mediate the tonic inhibitory control that dopamine hormone (GH) axis and control of body size, a differential phe- (DA) exerts on prolactin (PRL) synthesis and release. The notype that is considerable in males physiological significance of this inhibitory control has been Drd2 ⫺/⫺ mice display a shortfall of pituitary somatotropes, re-duced GH and IGF-I serum levels, and are dwarfs The abnormally large lactotrope/somatotrope ratio Received Dec. 12, 2012; revised Jan. 24, 2013; accepted Feb. 22, 2013.
observed in Drd2 ⫺/⫺ mice is highly reminiscent of a similar phe- Author contributions: D.N., M.I.P.-M., E.P.B., G.M.L., R.C.C., D.M.G., M.P., I.G.T., D.B.-V., and M.R. designed nomenon observed in lactating females, in which suckling- research; D.N., M.I.P.-M., E.P.B., G.M.L., R.C.C., D.M.G., M.P., I.G.T., and M.R. performed research; M.J.L. and M.R.
contributed unpublished reagents/analytic tools; D.N., M.I.P.-M., E.P.B., G.M.L., R.C.C., D.M.G., M.P., I.G.T., D.B.-V., induced inhibition of tuberoinfundibular DA neurons reduces and M.R. analyzed data; D.N., M.I.P.-M., M.J.L., and M.R. wrote the paper.
lactotrope D2R stimulation and promotes differentiation of so- This work was supported in part by the Howard Hughes Medical Institute (International Research Scholar Grant matolactotrope precursors into functional lactotropes to M.R.), Tourette Syndrome Association (to M.R.), Universidad de Buenos Aires (to M.R.), Consejo Nacional deInvestigaciones Científicas y Te´cnicas (CONICET, Argentina, to D.B.-V.), and the Agencia Nacional de Promocio´n Therefore, it is conceivable that Científica y Tecnolo´gica (to M.R. and D.B.V.). D.N., M.I.P.-M., E.P.B., G.M.L., D.M.G., M.P., and I.G.T., were recipients pituitary D2Rs play a critical role in establishing the terminal of doctoral fellowships from CONICET. We thank the National Hormone and Pituitary Program (National Institute of differentiation ratio of these two cell types from their common Diabetes and Digestive and Kidney Diseases) and Dr. A.F. Parlow for mouse RIA kits and antibodies, Guillermo Lanuza somatolactotrope precursor and, for providing Ai14 mice, and Vanina Rodriguez and Irina García Sua´rez for excellent technical assistance.
*D.N. and M.I.P.-M. contributed equally to this work.
therefore, in shaping the GH axis.
The authors declare no competing financial interests.
An alternative hypothesis that may account for the somato- Correspondence should be addressed to Dr. Marcelo Rubinstein, Vuelta de Obligado 2490, Buenos Aires 1428, trope shortfall and dwarfism of Drd2 ⫺/⫺ mice is that lack of Argentina. E-mail: [email protected].
central D2Rs impairs growth hormone-releasing hormone Copyright 2013 the authors 0270-6474/13/335834-09$15.00/0 (GHRH) or somatostatin (SST) function. In fact, the phenotype Noaín, Pe´rez-Milla´n et al. • Central D2Rs in Body Growth and Social Dominance J. Neurosci., March 27, 2013 • 33(13):5834 –5842 • 5835
observed in Drd2 ⫺/⫺ mice is similar to that present in lit/lit mice taining 2% normal goat serum (NGS); rabbit anti-rat adrenocortico- that lack functional GHRH receptors tropic hormone (rACTH; NHPP, NIH) diluted 1:1000 in KPBS/0.3% and in mutant mice lacking Ghrh Triton X-100 containing 2% NGS; and rabbit anti-LH (NHPP, NIH) because these two mouse models have fewer somatotropes diluted 1:12,000 in KPBS/0.3% Triton X-100 containing 2% NGS. Sec- and display dwarfism.
tions were then washed twice in PBS for 20 min at room temperature andincubated for 2 h with a secondary antibody at room temperature. To In the present study, we sought to determine whether GH detect GH, ACTH, and LH, a goat anti-rabbit Alexa Fluor 488 (Invitro- insufficiency in Drd2 ⫺/⫺ mice was due to the lack of D2Rs in gen) secondary antibody diluted 1:1000 in KPBS/0.3% Triton X-100 was pituitary lactotropes or in the brain by conducting a functional used; to detect prolactin, a donkey anti-goat Alexa Fluor 488 secondary dissection strategy based on cell-specific Drd2 inactivation in antibody diluted 1:500 in KPBS/0.3% Triton X-100 was used. Sections conditional Drd2 mutant mice We developed were washed twice in KPBS for 20 min and mounted in Vectashield a strain of transgenic mice expressing cre from a mouse prolactin (Vector Laboratories). Pituitary sections of neuroDrd2KO mice and gene promoter, Tg(Prl-cre) 1Mrub, that in parallel with transgenic control littermates were incubated with rabbit anti-GH antibody mice expressing cre from a rat nestin promoter, Tg(Nes-cre) 1Kln/J (NHPP, NIH) diluted 1:500 in KPBS/0.3% Triton X-100 containing 2% NGS. After washing twice for 20 min with KPBS, sections were incubated eliminate D2Rs selectively from pituitary lactotropes or from with donkey anti-rabbit FITC-coupled IgG (Santa Cruz Biotechnology)diluted 1:500 in KPBS/0.3% Triton X-100 for 2 h at room temperature.
cells of neural origin, respectively. Our results showed that al- After washing twice in KPBS, sections were incubated with propidium though male mice lacking D2Rs in pituitary lactotropes iodide to stain cell nuclei. Finally, sections were rinsed in KPBS twice for (lacDrd2KO) developed a normal GH axis and body size, those 10 min, mounted in water, and coverslipped using Vectashield (Vector lacking D2Rs in the brain (neuroDrd2KO) showed an impaired Laboratories). Confocal microscopy was performed to assess the number GH axis and reduced body growth. Deficits in the male pattern of of cells expressing GH per pituitary.
GH release are associated with feminization of sexually dimor- In situ hybridization. Brains and pituitaries were freshly removed and phic liver proteins, including major urinary proteins (MUP; frozen at ⫺40°C in isopentane, equilibrated at ⫺20°C in Tissue-Tek, a group of pheromones excreted at high levels in the cryosectioned at 16 ␮m, and mounted on SuperFrost slides (Fisher Sci- male urine and used in male-to-male social interactions entific), and stored at ⫺70°C until use. Sections were thawed and main- Therefore, in this study, we also sought to tained at room temperature for 30 min and then fixed in 4% investigate whether neuroDrd2KO male mice exhibited deficits paraformaldehyde in PBS for 30 min. After two washes in PBS, slideswere incubated in 0.1 M triethanolamine, pH 8, for 3 min, followed by in MUP levels and if their urine failed to induce territorial and incubation with triethanolamine-acetic anhydride 0.0025% for 10 min.
aggressive behaviors in conspecific males.
Slides were then rinsed twice in 2 ⫻ SSC (0.3 M NaCl, 0.03 M sodiumcitrate, pH 7.2), and, finally, dehydrated quickly in ascending ethanol Materials and Methods
concentrations. Sections were incubated with hybridization buffer (66% Generation of mice lacking D2Rs in pituitary lactotropes. A transgene construct formamide, 260 mM NaCl, 1.3 ⫻ Denhardt's) for 1 h at 57°C and hybrid- carrying 2.9 kb of 5⬘ mouse Prl promoter sequence driving cre recombinase ization started after adding 5 ⫻ 10 6 to 10 7 cpm/ml of an antisense mouse expression with a coding sequence for a nuclear localization was used to Drd2 exon 2 [ 35S]-riboprobe and proceeded overnight at 57°C in a wet generate Prl-cre transgenic mice. The 4.2 kb Prl-cre transgene was microin- chamber. The next day, slides were washed 4 times with 4 ⫻ SSC and then jected into the pronuclei of fertilized B6D2F2 eggs. The selected line incubated with RNase A (20 ␮g/ml in 0.5 M NaCl; 10 mM Tris-HCl pH 8; Tg(Prl-cre) 1Mrub was backcrossed for five generations to C57BL/6J.
1 mM EDTA pH 8) at 37°C for 30 min. Slides underwent consecutive Tg(Prl-cre) 1Mrub (n ⫽ 5) mice were consecutively crossed to Drd2loxP/loxP washes with 2⫻, 1⫻, and 0.5 ⫻ SSC/1 mM DTT and a final wash with mice to generate Drd2 loxP/loxP.Tg(Prl-cre) mice, named 0.1 ⫻ SSC/1 mM DTT at 65°C for 30 min. Finally, sections were dehy- lacDrd2KO. Transgenic mice were produced at the Instituto de Investigacio- drated in ascending ethanol concentrations, covered in Kodak N-TB2 nes en Ingeniería Gene´tica y Biología Molecular (CONICET, Argentina) and emulsion, and exposed for 15–20 d in a dark chamber until development animal procedures were conducted in accordance with local regulations and for posterior analysis. For the digoxigenin-labeled probe, prehybridiza- the Guide for the Care and Use of Laboratory Animals (National Institutes of tion was performed in a solution containing formamide 66%, NaCl 260 Health [NIH]).
mM, 1.3 ⫻ Denhardt's, EDTA 1.3 mM, Tris-HCl 13 mM, pH 8.0, and Generation of mice lacking D2Rs in neurons. To ablate D2Rs from dextran sulfate 13% at 57°C for 1 h. The probe was incubated at 65°C for cells of neural origin, Drd2 loxP/loxP mice were crossed to B6.Cg- 2 min in 0.5 mg/ml tRNA and 10 mM dithiothreitol and added to the Tg(Nes-cre) 1Kln/J (The Jackson Laboratory) to obtain cohorts of prehybridization buffer at a final concentration of 85 ng/ml. Hybridiza- Drd2 loxP/loxP (control) and Drd2 loxP/loxP.B6.Cg-Tg(Nes-cre) 1Kln/J tion was performed at 70°C overnight. After hybridization, the sections were washed with 0.2% SSC at 72°C for 1 h and then twice with a solution Immunofluorescence. Double transgenic mice, obtained by crossing containing 100 mM NaCl, 0.1% Triton X-100, and 100 mM Tris-HCl, pH Tg(Prl-cre) 1Mrub mice with the cre reporter mouse line Ai14 7.5. Sections were then incubated with the same solution plus 10% NGS received cabergoline (Laboratorios Beta) 0.5 mg/kg intraperi- for 4 h and then incubated with alkaline phosphatase-conjugated anti- toneally to increase prolactin signal and 2 h later were perfused with digoxigenin Fab fragments antibody (1:3500; Roche) at 4°C overnight.
paraformaldehyde 4% in phosphate buffered saline (PBS). Pituitaries The following day, samples were washed 5 times for 15 min each in PBS were incubated in perfusion solution at 4°C overnight, then in PBS for and then washed 4 times in a solution containing 100 mM NaCl, 50 mM 24 h, and finally in sucrose 10% in PBS for additional 24 h. Fixed pitu- MgCl , 0.1% Tween 20, and 100 m M Tris-HCl, pH 9.5. Then samples itaries were mounted in gelatin cubes (gelatin 10%, sucrose 10% in PBS) were incubated with NBT/BCIP (Roche) in the above-mentioned buffer and stored at ⫺80°C until use. Coronal 12 ␮m sections were cut using a overnight at room temperature. Signal development was stopped with Leica CM1850 cryostat and mounted on Superfrost slides (Fisher Scien- PBS, coverslipped with Mowiol (polyvinyl alcohol, glycerol in Tris 0.2 M, tific). For prolactin and luteinizing hormone (LH) detection, an antigen pH 8.5) and photographed.
retrieval procedure was performed incubating the sections in sodium Binding autoradiography. Brain and pituitary sections were obtained as citrate buffer 10 mM, pH 8.5, at 80°C for 10 min. Incubation with primary described for in situ hybridization, except they were stored at ⫺20°C antibodies were performed overnight at 4°C. The primary antibodies until use. Slides were thawed and maintained at room temperature for 30 used were as follows: goat anti-prolactin (sc-7805; Santa Cruz Biotech- min, and then preincubated in binding buffer (50 mM Tris-HCl, 120 mM nology) diluted 1:200 in KPBS/0.3% Triton X-100 containing 2% normal NaCl, 5 mM KCl, 2 mM CaCl , 1 m , pH 7.4) for 1 h. Next, slides donkey antiserum; rabbit anti-hGH (National Hormone and Pituitary were incubated for 1 h in binding buffer containing 1 nM [ 3H]- Program [NHPP], NIH) diluted 1:1000 in KPBS/0.3% Triton X-100 con- nemonapride. Nonspecific binding was determined in the presence of 10 5836 • J. Neurosci., March 27, 2013 • 33(13):5834 –5842
Noaín, Pe´rez-Milla´n et al. • Central D2Rs in Body Growth and Social Dominance ␮M S-(-)-sulpiride, and then sections were rinsed twice for 5 min in cold immobility for each mouse over the five trials. The tube test, performed water, completely dried with cold air, and exposed for 15–30 d to [ 3H]- to assess social dominance, consisted of introducing two adult mice in sensitive film (BIOMAX MR Scientific Imaging Film; Kodak) at room opposite entrances of a cylindrical acrylic tube of 3.4 cm diameter and 30 temperature in a dark cassette.
cm length and allowing them to interact in the center of the tube by Radioimmunoassays. Anterior pituitaries were dissected and homoge- removing a gate placed at 15 cm from each entrance. It is expected that nized in 0.2 ml of PBS. Protein contents were measured with the QUBIT one of the animals will force the other to retreat from the tube by pushing Fluorometer and the QUANT-IT protein Assay Kit (Invitrogen). Ali- it out. Successful trials were considered those in which a mouse retreated quots of equal quantities of protein were used to measure GH content by from the tube being pushed by the victor. We also observed atypical trials RIA. Serum PRL and GH pituitary content were measured by RIA using in which a mouse walked backwards by itself, not being forced by its kits provided by the National Institute of Diabetes and Digestive and opponent. Trials in which neither of the two mice pushed the other out Kidney Diseases (NIDDK; Dr. A.F. Parlow, NHPP, Torrance, CA). Re- during the 10 min test were considered null and discarded from the sults are expressed in terms of mouse PRL standard RP3 or mouse GH analysis. We assayed give pairs per genotype: C57BL/6J versus C57BL/6J standard AFP-10783B, respectively. Intra-assay and interassay coeffi- females, Drd2 loxP/loxP males, or neuroDrd2KO males. First, we trained all cients of variation were 7.2% and 12.8%, and 8.4% and 13.2%, for pro- mice to enter the tube by both entrances. After training, each pair of lactin and GH, respectively. For IGF-1 RIA, serum samples (15 ␮l) and confronting mice was assayed three times in the tube and the 15 trials IGF-1 standards were subjected to the acid-ethanol cryoprecipitation considered in the analysis. A variant of the intruder-resident aggression method. IGF-1 was determined using antibody (UB2– 495) provided by test, using castrated wild-type (WT) mice as intruders, was used to deter- Drs. L. Underwood and J.J. Van Wyk, and distributed by the Hormone mine the aggressive display promoted by the fresh urine of Drd2 loxP/loxP or Distribution Program of the NIDDK. Recombinant human IGF-1 (rh neuroDrd2KO male mice on isolated sex- and age-matched C57BL/6J IGF-1; Chiron) was used for radioligand and unlabeled standards. The mice C57BL/6J adult male mice were isolated for assay sensitivity was 6 pg per tube. Intra-assay and interassay coefficients at least 1 month and then wild-type castrated adult male mice were of variation were 8.2% and 14.1%, respectively. For testosterone RIA, an swabbed on their genitals and back with 150 ␮l of saline or fresh active testosterone RIA kit (DSL-4000; Diagnostic System Laboratories) collected urine from Drd2 loxP/loxP or neuroDrd2KOmice. After 1 min of drying, intruders were introduced to the home cage of an isolated Urine dosage of major urinary proteins. Urine was collected from male for 3 min. All tests were videotaped and analyzed for determi- 6-month-old mice between 1500 and 1700 h and centrifuged briefly for 3 nation of latency to first aggressive contact, consisting of kicking, min at 8800 ⫻ g. Two microliters of the supernatant was boiled in SDS biting, wrestling, or tumbling and total time spent attacking. The buffer. Samples were fractionated in 12% SDS-PAGE and subsequently odor-stimulated scent-marking test was performed using as experi- stained with Coomassie blue. The 20 KDa MUP represents the major mental subjects C57BL/6J adult male mice and, as stimulating odor protein component of mouse urine.
sources, the cages in which Drd2 loxP/loxP or neuroDrd2KO mice had Semiquantitative real-time PCR. Hypothalami from Drd2 loxP/loxP and been housed for the previous 48 h (test cages). The bedding of the test neuroDrd2KO mice were excised to determine Ghrh and Sst mRNA cages was removed but the cage itself was not cleaned. A rectangle (13 levels. Tissue samples were immediately homogenized in TRIzol reagent cm ⫻ 22 cm) of absorbent paper (Whatman, 3 mm Chr) was placed (Invitrogen), RNA extracted, and first strand cDNA synthesized using over the floor of each cage. Immediately, an adult male C57BL/6J and MMLV reverse transcriptase (Epicenter Biotechnolo- previously isolated for 1 month was placed in each test cage. The gies). Sense and antisense oligonucleotide primers were designed using subjects were allowed to explore the test cages for 15 min and then PrimerExpress software (Applied Biosystems). The sequences of the removed. The whole procedure took place during the first half hour of primers were as follows: Ghrh sense: 5⬘-GCCATCTTCACCACCAA-3⬘; the dark period in the behavioral room to profit from the enhanced Ghrh antisense: 5⬘-CCTCCTGCTTGTTCATGATGT-3⬘; Sst sense: 5⬘-TC activity of the animals in that period. The papers were then removed TGCATCGTCCTGGCTTT-3⬘; Sst antisense: 5⬘-CTTGGCCAGTTCCT from the test cages and photographed under UV light (UVP GTTTCC-3⬘; cyclophilin sense: 5⬘-GTGGCAAGATCGAAGTGGAGAA White/UV Transilluminator). Finally, the number of urine spots de- AC-3⬘; cyclophilin antisense 5⬘-TAAAAATCAGGCCTGTGGAATGTG- posited on each paper was counted manually, assisted by Photoshop 3⬘. Semiquantitative determination of Ghrh, Sst, and cyclophilin cDNA 7.0. Spots smaller than 1 mm or dragging marks were not considered were performed by kinetic PCR using 9.4 ␮l of TAQurate GREEN Real- in the analysis.
Time PCR MasterMix (Epicenter Biotechnologies) containing 100 ng of Statistics. One- or two-way ANOVA, repeated-measures ANOVA, cDNA, 0.4 ␮M primers, 10 mM Tris-HCl, 50 mM KCl, 3 mM MgCl , 0.2 ANOVA on ranks (Kruskal-Wallis on ranks), Student's test, one-tailed M deoxy-NTP, and 1.25 U Taq polymerase in a final volume of 10 ␮l.
analysis, and ␹ 2 2 ⫻ 2 contingency tables were applied as appropriate.
After denaturation at 95°C for 10 min, the cDNA products were ampli- Fisher LSD or Dunnett's contrasts were analyzed post hoc. In all experi- fied with 40 cycles, each cycle consisting of denaturation at 95°C for 30 s, ments, data are represented as means ⫾ SEM.
annealing at 61°C for 1 min, and extension at 72°C for 30 s. The accumu-lating DNA products were monitored by the ABI 7500 sequence detec- tion system (Applied Biosystems) and data were stored continuously Mice lacking D2Rs in pituitary lactotropes display normal
during the reaction. The calculations of the initial mRNA copy numbersin each sample were made according to the cycle threshold (CT) method body growth curves
as described previously The relative Ghrh To inactivate Drd2 expression in pituitary lactotropes, we first or Sst gene expression was normalized to that of the cyclophilin house- generated transgenic mice carrying 2.9 kb of 5⬘ flanking se- keeping gene using the standard curve method. Results are expressed as quences of the mouse prolactin gene ligated upstream of nuclear- arbitrary units (AU) for comparison among samples. AU was defined as targeted cre recombinase coding sequences. A selected pedigree, the expression level relative to a sample of wild-type mice (calibrator Tg(Prl-cre) 1Mrub, expressed functional cre in most PRL- producing cells of the anterior pituitary in a highly selective Food intake. Determination of food intake was performed in isolated manner as determined by immunofluorescence performed on adult Drd2 loxP/loxP or neuroDrd2KO mice. Animals and food (regular coronal pituitary sections of double transgenic mice obtained by chow) were weighed daily at the same hour during the light cycle (15:00 crossing Tg(Prl-cre) 1Mrub mice with the cre reporter mouse line Ai14 that expresses the fluorescent protein Behavioral testing. Haloperidol-induced catatonia was determined us- ing the horizontal bar test in which the time of immobility after 30 min of td-tomato upon cre recombination ). LacDrd2KO mice a single injection of saline or 1.5 mg/kg haloperidol (Tocris Bioscience) were obtained by successively breeding Tg(Prl-cre) 1Mrub mice was assessed. Five trials were performed for each animal with a 180 s with Drd2 loxP/loxP mice In situ hybridization cutoff for each trial. For the analysis, we considered the maximum time of analysis using a Drd2 exon 2 antisense riboprobe showed that

Noaín, Pe´rez-Milla´n et al. • Central D2Rs in Body Growth and Social Dominance J. Neurosci., March 27, 2013 • 33(13):5834 –5842 • 5837
lacDrd2KO mice of both genders, in con-trast to the effect observed in Drd2loxP/loxPcontrol mice, demonstrating the total ab-sence of functional D2Rs in lactotropes ofconditional lacDrd2KO mice ). Wealso found that haloperidol was equallyeffective lacDrd2KO and Drd2loxP/loxP control mice,in contrast to Drd2⫺/⫺ mice, which wereinsensitive to the D2R blocker's effect ), indicating that central D2R function isintact in lacDrd2KO mice. Therefore,lacDrd2KO mice are authentic lactotropeD2R-deficient mice. Body size and bodyweight curves of lacDrd2KO male micewere normal and identical to those observedin Drd2loxP/loxP male siblings ,G), aswell as GH axis parameters such as pituitaryGH and serum IGF-I levels, which were alsonormal in lacDrd2KO males ).
These results indicate that the absence ofD2Rs in pituitary lactotropes is not likely toaccount for the dwarfism found in Drd2⫺/⫺male mice.
NeuroDrd2KO mice have reduced body
weight and size
Transgenic mice expressing cre from a rat
nestin promoter were used in
combination with Drd2 loxP/loxP mice to
inactivate Drd2 alleles in neural progeni-
tors. As a result, neuroDrd2KO mice carry
null Drd2 alleles in cells of neural origin
and normally express Drd2 in other cell
types, as demonstrated at the molecular
and functional levels In situ
hybridization assays using a Drd2 exon 2
antisense riboprobe ) and [ 3H]-
nemonapride binding autoradiography
Figure 1.
LacDrd2KO mice display hyperprolactinemia and normal body growth. A, Cre-mediated td-tomato expression in
) revealed the absence of Drd2 various pituitary cell types. Immunofluorescence using primary antisera for PRL, GH, ACTH, and LH (green) performed on coronal mRNA and D2Rs, respectively, on coronal pituitary sections of Tg(Prl-cre) 1Mrub mice crossed with the cre inducible td-tomato line Ai14 (first column). Identical sections show brain sections taken from neuroDrd2KO td-tomato fluorescence (red, second column). Superimposed images show coexpression of cre and PRL but not GH (with a few
exceptions, see white arrow), ACTH or LH at lower (third column) and higher magnification (forth column). B, In situ hybridization
mice. NeuroDrd2KO mice failed to show using a Drd2 exon 2 antisense riboprobe in pituitary and brain sections of lacDrd2KO. Shown are whole pituitary sections and higher haloperidol-induced catatonia, in con- magnification of the anterior lobe and coronal brain sections (C) at the level of striatum and higher magnification. D, Serum PRL
trast to Drd2 loxP/loxP mice, but similar to levels in Drd2 loxP/loxP (n ⫽ 10) and lacDrd2KO (n ⫽ 11) females receiving saline (-) or haloperidol 3 mg/kg intraperitoneally (HAL, Drd2 ⫺/⫺ mice ), indicating the ⫹). Two-way ANOVA *p ⬍ 0.01 versus saline-treated Drd2loxP/loxP mice. E, Haloperidol (1.5 mg/kg, i.p.)-induced catatonia.
absence of functional central D2Rs. In ad- Latency to remove the front paws from an elevated horizontal bar was evaluated in Drd2 loxP/loxP (n ⫽ 4), lacDrd2KO (n ⫽ 5), and dition, D2R levels in the pituitary glands Drd2 ⫺/⫺ (n ⫽ 3) male mice. *p ⬍ 0.01 versus saline for each genotype. F, Nose to anus length of neuroDrd2KO and Drd2 loxP/loxP
of neuroDrd2KO mice appeared to be male littermates. G, Body weight curves of Drd2 loxP/loxP and lacDrd2KO male littermates. No significant differences were found
similar to those observed in Drd2 loxP/loxP between genotypes. H, Radioimmunoassay showed normal pituitary GH and serum IGF-I levels in lacDrd2KO mice relative to their
mice , B) and functionally nor- Drd2 loxP/loxP littermates. Bars and circles represent the mean ⫾ SEM.
mal, as evidenced by the normal prolactinlevels observed in neuroDrd2KO mice lacDrd2KO mice lacked D2Rs in pituitary lactotropes while re- ) and the increase observed after receiving a 3 mg/kg taining normal Drd2 expression in pituitary melanotropes of the intraperitoneal haloperidol injection ). Therefore, intermediate lobe ) and in all brain areas examined neuroDrd2KO mice lack D2Rs in the CNS but retain normally ). To investigate whether ablation of D2R in lacDrd2KO mice functional D2Rs in the anterior pituitary. NeuroDrd2KO male was complete, we measured basal serum PRL levels and, more mice showed reduced body weight ) and length ) importantly, the acute effect of the D2R blocker haloperidol.
compared with their Drd2loxP/loxP male littermates. Body weight of LacDrd2KO mice displayed hyperprolactinemia that was par- neuroDrd2KO mice was 14.2% lower than Drd2loxP/loxP at 2 months ticularly significant in females ). The D2R antagonist halo- of age and this difference increased over time, being 21.7% lower at 6 peridol (3 mg/kg, i.p.) failed to increase serum PRL levels in months ). Body size of neuroDrd2KO mice at weaning was

5838 • J. Neurosci., March 27, 2013 • 33(13):5834 –5842
Noaín, Pe´rez-Milla´n et al. • Central D2Rs in Body Growth and Social Dominance Figure 2.
NeuroDrd2KO mice have reduced body weight and size. A, B, In situ hybridization
assay using a Drd2 exon 2 [ 35S]-UTP antisense riboprobe (A) and [ 3H]-nemonapride binding
autoradiography (B) in brain and pituitary coronal sections of Drd2 loxP/loxP and neuroDrd2KO
mice. Sections of Drd2 ⫺/⫺ mice were included as negative controls. C, Haloperidol (HAL, 1.5
mg/kg, i.p.)-induced catatonia. Latency to remove the front paws from an elevated horizontal
bar was evaluated in neuroDrd2KO mice and compared with Drd2 loxP/loxP (n ⫽ 6) and
Drd2 ⫺/⫺ (n ⫽ 6) mice. *p ⬍ 0.01 haloperidol versus saline for each genotype. D, Serum PRL
Figure 3.
NeuroDrd2KO mice display an impaired GH axis. A, Immunofluorescence using an
levels of female mice (n ⫽ 10 –12) receiving haloperidol (HAL, 3 mg/kg, i.p.) or saline. *p ⬍ anti-hGH primary antiserum performed on 4 ␮m pituitary slices of Drd2 loxP/loxP (n ⫽ 3) and 0.05 haloperidol versus saline for each genotype. E, Body weight curves of neuroDrd2KO and
neuroDrd2KO (n ⫽ 3) mice showed fewer pituitary somatotropes (green) in neuroDrd2KO mice Drd2 loxP/loxP male mice (n ⫽ 7–12). Repeated-measures ANOVA genotype: F relative to control mice. Cell nuclei were stained with propidium iodide (red). B, Right, Percent-
p ⬍ 0.005. *p ⬍ 0.01 neuroDrd2KO versus Drd2 loxP/loxP littermates, post hoc Fisher analysis. F,
age of GH-positive cells, one-way ANOVA (OWA): F ⫽ 18.01, *p ⬍ 0.02. Left, radioimmu- Nose to anus length of neuroDrd2KO and Drd2 loxP/loxP male mice (n ⫽ 9 –11) measured 3, 6, noassay of GH concentration in anterior pituitary glands of Drd2 loxP/loxP (n ⫽ 7) and and 9 weeks after birth. Repeated-measures ANOVA genotype: F ⫽ 21.35, p ⬍ 0.005.
neuroDrd2KO (n ⫽ 10) siblings (OWA: F ⫽ 7.54, *p ⬍ 0.05). C, Right, relative hypotha-
*p ⬍ 0.05 for neuroDrd2KO versus age-matched Drd2 loxP/loxP littermates, post hoc Fisher anal- lamic mRNA levels of Ghrh in Drd2 loxP/loxP (n ⫽ 9) and neuroDrd2KO (n ⫽ 9) mice determined ysis. G, Food consumption was measured for 7 consecutive days in adult neuroDrd2KO and
by semiquantitative real-time PCR (OWA: F ⫽ 6.32, *p ⬍ 0.05). Left, relative hypotha- Drd2 loxP/loxP (n ⫽ 5–10) mice. Averaged food intake (left) and food intake relative to animal lamic mRNA levels of Sst from Drd2 loxP/loxP and neuroDrd2KO mice (n ⫽ 13). Samples were weight (right) is plotted. Bars and circles represent the mean ⫾ SEM.
normalized to cyclophilin mRNA. Bars denote the mean ⫾ SEM. All mice were 6 –9 months old.
the nervous system develop an immature GH axis that limits the already significantly lower (10.2%) than control littermates and the body growth program.
difference persisted until reaching adulthood ). The de-creased growth exhibited by neuroDrd2KO male mice was not NeuroDrd2KO mice have low levels of the GH-dependent
due to reduced feeding, because the daily food intakes of liver proteins IGF-I and MUP
neuroDrd2KO and Drd2loxP/loxP siblings were similar ).
To determine whether the impaired GH axis found in Therefore, these results demonstrate that the sole absence of D2Rs in neuroDrd2KO mice has downstream consequences in GH- the nervous system impairs normal growth rates and produces dependent liver hormones, we measured IGF-I and MUP levels.
smaller mice.
Serum IGF-I levels were 18.7% lower in neuroDrd2KO malemice compared with those found in their control siblings NeuroDrd2KO mice have an impaired GH axis
), whereas MUP levels were markedly reduced (45.6%) in the We analyzed the relative number of somatotropes and GH con- urine of neuroDrd2KO male mice ). Interestingly, MUP tent in pituitaries from neuroDrd2KO and control male mice.
levels of the male mutants were similar to those found in the urine Immunofluorescence performed on pituitary slices taken from of their Drd2 loxP/loxP female littermates ). In contrast, neuroDrd2KO male mice showed a 28.1% shortfall of GH- serum testosterone levels in neuroDrd2KO male mice were sim- positive cells compared with control male mice , B). In ilar to those found in control male littermates, in clear contrast to agreement, a great reduction (63.2%) in GH content was deter- testosterone levels measured in adult wild-type females and cas- mined by radioimmunoassay in pituitaries from neuroDrd2KO trated wild-type males ). These results suggest that the mice ). Analysis of the hypothalamic content of Ghrh and reduced MUP levels were primarily the consequence of an im- Sst mRNAs using a semiquantitative RT-PCR assay showed that paired GH axis.
neuroDrd2KO males had 30% lower Ghrh mRNA levels com-pared with their Drd2 loxP/loxP littermates ), whereas hy- Central D2Rs, social dominance, and territorial behavior
pothalamic Sst mRNA levels were not significantly altered The higher MUP content typically found in the urine of adult ). These results demonstrate that male mice lacking D2Rs in males is thought to promote a variety of male-specific behaviors

Noaín, Pe´rez-Milla´n et al. • Central D2Rs in Body Growth and Social Dominance J. Neurosci., March 27, 2013 • 33(13):5834 –5842 • 5839
Figure 4.
NeuroDrd2KOmice have reduced IGF-I and MUPs and normal testostene levels. A,
Radioimmunoassay of serum IGF-I levels in neuroDrd2KO (n ⫽ 14) and Drd2 loxP/loxP (n ⫽ 20)mice (one-way ANOVA [OWA]: F from male Drd2 loxP/loxP (n ⫽ 17) and neuroDrd2KO (n ⫽ 12) adult mice and from theirDrd2 loxP/loxP (n ⫽ 16) and neuroDrd2KO (n ⫽ 9) female littermates. Two-way ANOVA showedsignificant main effects both for sex and genotype (sex effect: F ⫽ 26.68, *p ⬍ 0.00001 and genotype effect: (1,50) ⫽ 17.84, *p ⬍ 0.0005). C, Serum testosterone levels determined
by radioimmunoassay in neuroDrd2KO male mice (n ⫽ 9) compared with their Drd2 loxP/loxP
male littermates (n ⫽ 9). Castrated wild-type adult male mice (n ⫽ 4) and wild-type adult
females (n ⫽ 5) were included as negative controls (OWA: F
⫽ 5.0673, p ⬍ 0.008). *p ⬍ 0.05 for wild-type castrated males and wild-type females versus Drd2 loxP/loxP and neuroDrd2KOmale mice, post hoc Fisher's LSD. Bars represent the mean ⫾ SEM.
that are critical for reproductive fitness, such as social recogni-tion, territorial scent-marking, and aggression To evaluate whether the feminized lowerMUP levels found in the urine of neuroDrd2KO male mice mod-ify these evolutionarily conserved behaviors, we designed a seriesof behavioral tests. First, we examined how adult male C57BL/6JWT mice reacted when confronted with either Drd2 loxP/loxP orneuroDrd2KO male adults in a social dominance experimentalparadigm called the double entrance tube test Normally in this test, when two adult male mice are placed at theopposite extremes of a cylindrical tube, they both gain access tothe center and, after close mutual inspection, one moves forward,forcing the other to walk backwards until stepping down from the Figure 5.
Urine from adult neuroDrd2KO males fails to promote social and territorial behav- end of the tube , right). However, when confronting fe- iors. A, Left, C57BL/6J adult females, Drd2 loxP/loxP adult males and neuroDrd2KO adult males
males, adult male mice do not show this behavior and back out were challenged with C57BL/6J adult males in a social-dominance tube test. Fifteen trials were spontaneously without showing signs of social dominance. When performed for each genotype. The behavior of C57BL/6J males was significantly different when we challenged C57BL/6J adult males with sex- and age-matched confronting C57BL/6J females compared with Drd2 loxP/loxP males (one-tailed ␹ 2 *p ⬍ 0.05).
Drd2 loxP/loxP mice, we observed a similar number of retreats in C57BL/6J males confronting neuroDrd2KO males showed an intermediate phenotype because mice of each genotype: C57BL/6J males backed out from the tube their behavior was not different from that observed when confronting either C57BL/6J females in seven of 15 trials, whereas Drd2 loxP/loxP males backed out in the or Drd2 loxP/loxP males (one-tailed ␹ 2 p ⬎ 0.05). Right, Representative images of a typical test:(1) two mice approaching the center of the tube, (2) mutual inspection, and (3) the mouse on other eight ). Conversely, when challenged with WT fe- the right pushes the other out. B, Intruder-resident test. Isolated C57BL/6J adult male residents
males, C57BL/6J adult males backed out of the tube in 12 of 15 (n ⫽ 18) were challenged with C57BL/6J castrated males swabbed on their backs and genital encounters without showing social dominance behavior areas with saline (n ⫽ 6), urine from Drd2 loxP/loxP adult male mice (n ⫽ 6), or urine from ). Interestingly, C57BL/6J adult male mice challenged against neuroDrd2KO adult males (n ⫽ 6). Left, latency to the first resident's attack (one-way ANOVA sex- and age-matched neuroDrd2KO mice exhibited behavioral ⫽ 4.55, p ⬍ 0.05). *p ⬍ 0.05 compared with saline, post hoc Fisher analysis.
responses similar to those shown when challenged against Right, total time spent attacking (Kruskal-Wallis OWA on ranks: H ⫽ 6.65 with 2 degrees of C57BL/6J females. In this case, C57BL/6J males backed out of the freedom, p ⬍ 0.05). *p ⬍ 0.05 compared with saline, post hoc Dunnett's comparison. C, Scent
tube spontaneously in 10 of 15 encounters and moved forward marking test. Left, C57BL/6J adult male mice were challenged in a cage previously occupied by neuroDrd2KO males in only five trials ). The different a Drd2flox/flox (n ⫽ 5) or a neuroDrd2KO (n ⫽ 8) adult male mouse. Urine marks made by the reaction of C57BL/6J adult males toward neuroDrd2KO adult visitor during 5 min were counted under UV light (OWA: F 14.66, *p ⬍ 0.005). Right, two representative examples of spotted urine are shown. Bars represent the mean ⫾ SEM.
males compared with that observed when confronting theirDrd2 loxP/loxP littermates could be due, at least in part, to the rel-atively smaller size of neuroDrd2KO mice and/or to their lower of the intruder, resident mice exhibited aggressive behavior that concentration of urinary MUP. To further investigate this lat- varied in latency and intensity depending on the nature of the ter hypothesis, we tested the ability of the urine taken from liquid swabbed on the intruder. Latency to the first attack was Drd2 loxP/loxP and neuroDrd2KO male adults to promote territo- 65 ⫾ 17 s when castrated mice received urine from Drd2loxP/loxP rial male-to-male aggression in an adapted version of the mice, and this value increased significantly when mice were intruder-resident aggression test To swabbed with either saline or urine taken from neuroDrd2KO this end, each C57BL/6J adult male resident mouse reared in male adult mice , left). Moreover, 3 of 6 intruders isolation for 1 month was challenged with a castrated adult WT swabbed with saline and 3 of 6 intruders swabbed with male intruder mouse placed into the resident's cage. Each cas- neuroDrd2KO male mice urine received no attacks from the res- trated mouse was swabbed on its back and genital area with ident males during the 3 min test. Therefore, when resident males saline or freshly collected urine taken from Drd2 loxP/loxP or were confronted with castrated males swabbed with urine taken neuroDrd2KO adult male mice. Upon close olfactory inspection from neuroDrd2KO mice, the latency to the first attack increased 5840 • J. Neurosci., March 27, 2013 • 33(13):5834 –5842
Noaín, Pe´rez-Milla´n et al. • Central D2Rs in Body Growth and Social Dominance markedly and the time spent attacking decreased compared with neurons of the indirect extrapyramidal pathway castrated intruders swabbed with urine taken from Drd2 loxP/loxP because these two conditional Drd2 knock-out models male adults , right). Finally, we performed a third exper- exhibited normal growth curves. Because D2Rs couple to inhib- iment to investigate whether C57BL/6J adult male mice displayed itory signals that diminish neuron activity, a candidate neuronal different scent-marking behavior when placed in a cage where a type is a group of periventricular SST neurons that make synaptic sex- and age-matched Drd2 loxP/loxP or a neuroDrd2KO mouse contacts with GHRH arcuate neurons to exert a tonic inhibition was previously housed. A stereotyped behavior commonly ob- of Ghrh expression and GHRH release served when a male mouse detects odorant cues from another and control the rhyth- adult conspecific male is the induction of widespread urine de- mic oscillations of GHRH neuronal firing that establish the male positions that are interpreted as increased scent-marking activity pattern of GH release to cover the area with its own scent This However, a semiquantitative RT-PCR determination only behavior is greatly reduced when the adult male is placed in a found a tendency for increased hypothalamic Sst mRNA levels location previously visited only by a castrated or a subordinate in neuroDrd2KO mice that never reached statistical signifi- male By measuring the number of urinary spots, we determined that C57BL/6J adult male mice The present study demonstrates that central D2Rs regulate a reared in isolation displayed greater scent-marking activity when neuroendocrine/exocrine cascade that controls body growth and placed in a cage previously occupied by a Drd2 loxP/loxP mouse participates in male-to-male territorial behavior. This multistep rather than by a neuroDrd2KO male adult littermate ).
signaling cascade initiates in brain centers that regulate the hypo- These results indicate that wild-type male adults do not exhibit thalamic level Ghrh expression and GHRH release, which then typical male-to-male social and territorial behavior when ex- relays to the pituitary to control the pattern of GH release. GH posed to neuroDrd2KO males or to their urine. Therefore, cen- surges from the pituitary of male rodents are characterized by tral D2Rs are necessary for the masculinization of the GH axis high-amplitude and low-frequency pulses intercalated among and, therefore, for the production of higher levels of pheromones long nadir periods, which permit the resensitization of GH recep- typically found in the urine of normal males.
tor signaling by a Janus kinase/signal transducer and activation oftranscription (JAK-STAT) complex that specifically involves STAT5a and STAT5b In this study, we conducted a differential cell-specific ablation Only when long interpulse strategy to eliminate D2Rs selectively from pituitary lactotropes nadirs of GH levels are attained in puberal males are high tran- and, independently, from brain neurons. Whereas lacDrd2KO scriptional rates of Igf1 and other somatomedins that promote mice displayed normal body growth, neuroDrd2KO mice body growth observed in the liver and extrahepatic sites showed deficits in the GH axis that led to reduced body weight The lower serum levels of IGF-I observed in and length, recapitulating the dwarf phenotype observed in neuroDrd2KO mice are consistent with their smaller body size Drd2 ⫺/⫺ mutants. Based on the analysis of lacDrd2KO mice, we and weight, a phenotype that in the wild could be detrimental for concluded that the absence of lactotrope D2R is not responsible reproductive fitness due to reduced competitiveness among for the altered lactotrope/somatotrope ratio previously observed males. The sexually dimorphic liver proteins that depend on the in Drd2 ⫺/⫺ mice, ruling out its participation in the termi- male pattern of GH release is manifold and include MUPs nal differentiation of these two cell types. Furthermore, This group of pheromones se- neuroDrd2KO mice showed fewer somatotropes, diminished pi- creted into the blood and then excreted in the urine are known to tuitary GH content, and lower levels of hypothalamic Ghrh ex- promote social recognition and territorial dominance between pression. Therefore, central D2Rs are essential for the normal males Therefore, an maturation of the GH axis and full development of the body emerging downstream finding disclosed by this study is that the growth plan. Selective ablation of D2Rs in the brain while main- urine of mice lacking D2Rs in the nervous system showed a lower taining normal Drd2 expression in the pituitary was achieved by content of MUP and, therefore, lost the signaling cues that trigger using a strain of transgenic mice expressing cre from a nestin territorial behavior in other males. Typically, MUP levels in males promoter. A similar strategy has been used previously to elimi- are much higher than in females and this nate the transcription factor CREB from the CNS difference not only depends on the sexually dimorphic pattern because the nestin gene (Nes) is expressed in of GH pulsatility, but also on testosterone concentration, be- embryonic dividing cells derived from the neuroectoderm but cause castrated males have feminized MUP levels and females not from the oral ectoderm treated with testosterone display male MUP levels and is particularly absent from progenitors and differ- Because testosterone concentration was normal in entiated cells of all anterior pituitary cell lineages neuroDrd2KO mice, the reduced MUP levels are likely to be the In fact, neuroDrd2KO mice sole consequence of an impaired GH axis resulting from lack of showed normal Drd2 expression in the intermediate and anterior Drd2 expression in the CNS. MUPs are encoded by ⬎30 genes, lobe of the pituitary but complete absence of expression in the mainly synthetic, in a locus of mouse chromosome 4 CNS. The differential response to haloperidol-induced hyperp- The large allelic variation at this rolactinemia and catatonia observed in lacDrd2KO and locus and the relative amount of each MUP in the mouse urine neuroDrd2KO mice confirmed, at the functional level, our suc- contributes to generate a great combinatorial diversity of individ- cessful attempt to inactivate Drd2 expression selectively in the ual patterns that are believed to provide, in the wild, a unique pituitary or in the brain.
biological identity card for each conspecific Identifying the Drd2-expressing neurons in the CNS control- In fact, male mice use MUPs to scent-mark and counter- ling the maturation of the GH axis is still pending, although our mark territories The ability of MUPs own studies ruled out the participation of DA neurons expressing to create a chemical barrier for territorial defense and to promote D2 autoreceptors and striatal medium spiny territorial protection adds to the adaptive behavioral inventory Noaín, Pe´rez-Milla´n et al. • Central D2Rs in Body Growth and Social Dominance J. Neurosci., March 27, 2013 • 33(13):5834 –5842 • 5841
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