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Biochemical and Biophysical Research Communications 266, 556–559 (1999)

Article ID bbrc.1999.1851, available online at http://www.idealibrary.com on

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istribution of D5 Dopamine Receptor mRNAn Rat Ventromedial Hypothalamic Nucleus

an Zhou, Ede Marie Apostolakis, and Bert W. O’Malley1

epartment of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030

eceived November 11, 1999

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Estrogen induces lordosis through, in part, estrogeneceptor (ER)-mediated synthesis of progesterone re-eptors (PR) in the ventromedial nucleus (VMN). Initro, PR is activated by the neurotransmitter dopa-ine through D1-like receptors (1). In vivo, lordosis is

nduced by dopamine, an effect mediated in part byR and D5 dopamine receptors. The purpose of theresent study was to determine mRNA distribution of1-like receptors in the female rat brain using RT-CR combined with punchout microdissection tech-iques. Employing specific primers to D5 and D1 dopa-ine receptors, we found detectable expression levels

f D5 dopamine receptor mRNA in VMN as well as thercuate nucleus/median eminence (ArcN/ME). In con-rast, D1 dopamine receptor mRNA was detected onlyn VMN. By using this highly sensitive and specificT-PCR methodology, we have confirmed the pres-nce of D5 dopamine receptor mRNA in an area of therain that regulates reproductive behavior throughR. The data support the previous observation that D5

opamine receptors in VMN contribute to facilitationf female reproductive behavior by D1-like agonists.1999 Academic Press

As a major catacholamine of the brain, the neuro-ransmitter dopamine is localized predominately ineurons that are distributed in the substantia nigra,ars compacta, ventral tegmental area, and hypothal-mus. Dopaminergic neurons are organized into threeain pathways, known as nigrostriatal, mesolimbic,

nd tuberoinfundibular systems. These dopaminergicystems are involved in the regulation of a variety ofiological functions, such as locomotion, cognition, af-ect, and neuroendocrine secretion (3). We (2, 4) andthers (5) have shown that centrally administeredopamine and several D1-like agonists includingKF38393 facilitate the reproductive behavior lordosis

n estrogen-primed rats. Further, the induction of lor-

1 To whom correspondence should be addressed. Fax: (11)-713-90-1257. E-mail: berto@bcm.tmc.edu.

556006-291X/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

otic behavior by D1-like agonists can be blocked byhe antiprogestins RU486 and antisense oligonucleo-ides to PR administered into the third ventricle (4),uggesting a dependency of dopamine-induced lordosisn PR. In support of the hypothesis that the unoccu-ied PR is essential for dopamine induced lordotic be-avior, estrogen primed mice carrying a null mutationf PR also fail to exhibit lordosis following D1-liketimulation (6).Dopamine exerts its effects in the central and pe-

ipheral nervous systems through a family of geneti-ally distinct membrane-bound receptors that are gen-rally divided into 2 classes, D1-like and D2-likeeceptors. Each class of dopamine receptors transducests signals by coupling to guanine nucleotide-bindingegulatory protein (G protein, for review see 7). Theembers of the D1-like dopamine receptors—D1 (D1A),5 (D1B), D1C and D1D—stimulate adenylyl cyclase for

he formation of adenosine 39, 59-monophosphatecAMP) in response to agonists. In contrast, inhibitionf cAMP formation is a general property of D2-likeopamine receptors. Importantly, D1-like dopamine re-eptors mediate the in vitro transcriptional activationf PR and estrogen receptors (ER) by dopamine,KF38393 in absence of steroids (for review see 8, 9).retreatment of cells with the D1-like antagonistCH23390 blocks the effect of SKF38393. Thus, the initro findings are consistent with the aforementionedn vivo findings for reproductive behavior since lordosiss dependent on estrogen and ER-dependent transcrip-ion (for review see 10).

Reproductive behavior in female rodents is mediatedy the transcriptional activity of the classical ERa (10).vidence from pharmacologic, electrical and lesiontudies clearly implicate ER in the ventrolateral por-ion of the VMN in the performance of this behavior.

ithin 24–48 h after priming with 17b-estradiol (EB),RNA and protein for PR are detected in the ventro-

ateral VMN. Although not absolutely necessary for allNS pathways mediating sexual behavior (11), theppearance of PR in the VMN is closely correlated withhe exhibition of lordosis in EB-primed rats (12). Cer-

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Vol. 266, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ainly, the facilitatory function of P and several D1-likegonists is abolished when PR synthesis in the VMN isuppressed by PR antisense oligonucleotides (4) or inransgenetic mice (6). In addition to the VMN, facilita-ory processes from the ArcN/ME project to the VMN torovide additional modulation for the exhibition of re-roductive behavior (10). As with P, centrally admin-stered D1-like agonists fail to induce sex behavior inither the absence of PR (4, 6) or the D5 dopamineeceptor (2). Surprisingly, however, D5 dopamine re-eptors have not been detected by in situ hybridizationn the VMN or the ArcN/ME of EB-primed females (2),uggesting that the mRNA may be below the level ofensitivity for this technique. To test this hypothesis,e re-examined the distribution of D5 dopamine recep-

or in the rat hypothalamus using RT-PCR combinedith punchout microdissection technique. The present

esults demonstrate, for the first time, the expressionf D5 dopamine receptor mRNAs in the VMN of EB-rimed female rats. Further, we report the coexpres-ion of D5 and D1 dopamine receptor mRNAs in VMN.nlike the VMN, only D5 dopamine receptor mRNAsere found expressed in hypothalamic ArcN/MEunchouts by RT-PCR. Thus, the data confirm theresence of D5 dopamine receptor mRNA in an area ofhe brain that regulates reproductive behavior throughctivation of the PR and provide additional evidence toupport the hypothesis that ligand-independent acti-ation of steroid receptors can alter biological function.

ATERIALS AND METHODS

Animals. OVX female rats (160–200 g) were purchased fromharles River (Houston, TX) and maintained as described previously

2). EB primed animals (50 mg sc, at about 44 h before sacrificing)ere anesthetized with an anesthetic combination containing ket-mine, xylazine and acepromazine. After decapitation, the brainsere quickly removed, iced, and then blocked for slicing. The VMNnd ArcN/ME were punched out individually according to Paxinosnd Watson’s rat brain atlas (13) using borosilicate glass tubesWorld Precision Instruments, Inc., Sarasota, FL). The punchoutiopsies were snap-frozen in a dry-ice ethanol bath, followed by totalNA purification or stored at 280°C until use. All animal studiesere conducted in accordance with NIH guidelines and were ap-roved by the Institutional Animal Care and Use Committee.

Total RNA purification. Total RNA was extracted from the brainunchout biopsies with the High Pure RNA Tissue Kit (Boehringer-annheim, Mannheim, Germany) according to the manufacturer’s

nstruction. Briefly, the brain punchouts were homogenized withxtraction buffer (4.5 M guanidine-HCl, 100 mM sodium phosphate,H 6.6), followed by 1 min centrifugation at 13,000 3 g. The super-atants were transferred to new tubes and 0.5 volume ethanol wasdded. After mixing, lysates were aliquoted into High Pure filterubes and centrifuged at 13,000 3 g for 2 min. Filter columns werencubated with DNase incubation buffer (1 M NaCl, 20 mM Tris/HCl,0 mM MnCl2, pH 7.0) and RNase free amplify degree DNase-I (200nits) for 30 min at room temperature to digest the residual contam-

nating DNA. After DNase digestion, the filter tubes were centri-uged at 8,000 3 g for 30 sec and washed twice with wash bufferrovided in the kit. Final RNA products were eluted with 100 mluclease-free, sterile, double distilled water. The concentration and

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nd 280 nm (14).

Reverse transcription-polymerase chain reaction analysis. Thexpression of rat D5 and D1 dopamine receptor mRNA in the VMNnd ArcN/ME were determined using RT-PCR. First, each cytoplas-ic RNA sample (10 ml) was reverse transcripted with SuperScripreamplification System (Life Technologies, Inc., Gaithersburg, MD)

n a total volume of 20 ml using 0.5 mg of oligo(dT) for 50 min at 42°C.hen, the cDNA of D1 and D5 dopamine receptors were detectedsing specific primers.Rat D5 and D1 dopamine receptor mRNA specific primers and the

CR strategy employed here as previously described (15, 16). Prim-rs used for rat D5 dopamine receptor mRNA (GenBank Accessiono. M69118) detection were: 59-AGT CGT GGA GCC TAT GAA CCTAC-39 (nucleotides 1459–1518), and 59-GCG TCG TTG GAG AGATT GAG ACA-39 (nucleotides 2011–1988). The size of the amplified5 dopamine receptor cDNA was 517 bp. Primers used for rat D1

opamine receptor mRNA (GenBank Accession No. M35077) detec-ion were 59-CAG TCC ATG CCA AGA ATT GCC AGA-39 (nucleo-ides 762–786), and 59-AAT CGA TGC AGA ATG GCT GGG TCT-39nucleotides 987–963). The size of this D1 dopamine receptor ampli-on was 225 bp.Ten microliters of each cDNA sample were added to a PCR reac-

ion mix containing 4.0 mM MgCl2, 50 mM KCl, 10 mM Tris/HCl, 1.0M dNTPs, 20 pmol of each primer (for D5 or D1 dopamine receptor

ndividually) and 2.5 units Taq polymerase (Life Technology, Inc.) intotal volume of 50 ml. The reaction series were initiated on a

TC-100 programmable thermal controller (MJ Research, Inc., Wa-ertown, MA). Forty cycles of “touchdown” PCR amplification wereerformed using the following parameters: 94°C for 1 min, 58°C formin, 72°C for 3 min, for the first 20 cycles. After 20 cycles, the

nnealing temperature was decreased by 1°C every other cycle,esulting in a final annealing temperature of 48°C.PCR products were separated by electrophoresis in 1.5% agarose

el and the amplicons were visualized by staining with ethidiumromide. Representative products were cut off and purified with a gelxtraction kit (Qiagen, GmbH., Hilden, Germany). Purified PCRroducts were sequenced using a dye termination procedure by thehild Health Research Center Core at Baylor College of Medicine.he homology of amplified cDNAs to published sequences werehecked by BLAST homology searching program (National Centeror Biotechnology Information, U.S.A.).

Negative controls for contamination from extraneous and genomicNA were run in every individual experiment to ensure that neitherenomic nor extraneous DNAs contributed to the PCR products.xtraneous contamination was checked by replacing the cellular

emplates with water.

ESULTS AND DISCUSSION

In the present study, we have employed the highlyensitive and specific method of RT-PCR combinedith punchout microdissection technique to determine

he distribution of dopamine receptor mRNAs in theMN and ArcN/ME region of the female rat hypothal-mus after EB-priming. As molecular probes, we usedligonucleotide primers which have been used success-ully in the detection of D1-like receptor expression andtouchdown” PCR amplification. We report that a 517p amplicon with 100% homology to the D5 dopamineeceptor mRNA (17) was detected in the total RNArepared from the VMN of EB-primed, OVX rats (Fig., lane 2, 4). Also detected in the VMN punchouts was225 bp cDNA with 100% homology to the D1 dopa-

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ine receptor mRNA (18) (Fig. 1, lane 1, 3). In con-rast, the only cDNA detected in the ArcN/MEunchout was homologous with D5 mRNA. The dataupport the original findings (2) that D5 receptor in theMN plays a role in the induction of lordosis.In the past, in situ hybridization has been used ex-

ensively to determine the distribution of dopamineeceptors in the brain since there is a lack of selectiveompounds, precluding the possibility to stimulate orlock a single subset of D1-like receptors. D1-like re-eptors display an indistinguishable pharmacologicalrofile and are both coupled to Gsa transduction path-ays (18). In situ hybridization and Northern blotnalyses have revealed a distinctive quantitative andualitative distribution throughout the male brain. D1

opamine receptor mRNA is found in abundance in thetriatum, nucleus accumbens and olfactory tubercule19) whereas only minor amounts of D5 dopamine re-eptor mRNA is found in the hippocampus, reuniens,nd parafascicular nucleus of the thalamus, and hypo-halamus (17, 20). Such segregate patterns of localiza-ion of D5 and D1 receptors suggest that they exertistinct regulatory roles in different circuits for dopa-ine neurotransmission.The existence of D5 dopamine receptor mRNA in

eurons of the female rat VMN was first suggestedhen we found that antisense oligonucleotide against

he D5 dopamine receptor mRNA abolished the facili-atory effect of D1-like dopamine agonists on lordoticehavior (2). Initial in situ study for D5 dopamine re-eptor failed to detect the presence of the mRNA inMN and ArcN/ME area. As is suggested by theresent findings, this discrepancy may have been dueo expression levels below the level of sensitivity for initu hybridization. Alternately, cyclic changes in theeproductive physiology of female animals may regu-ate the expression level of D5 dopamine receptor,

FIG. 1. Distribution of D1 and D5 dopamine receptor mRNAs inentromedial nucleus, arcuate nucleus, and median eminence of ratrain measured by RT-PCR. Tissues of rat brain VMN and ArcN/MEere punched out and total RNA from these tissues were purified,

ollowed by RT-PCR detection as described. Twenty-five microlitersf PCR products were isolated using 1.5% agarose gel electrophore-is, and the DNA bands were visualized with ethiodium bromide (0.5g/ml). The pictures were captured with Alpha Imager 2000 docu-entation and analysis system. The position of molecular size mark-

rs is indicated to the left.

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imilar to the expression of PR, in specific dimorphicegions of the female brain. For example, it is alreadynown that estrogen modulates the central dopaminer-ic system through dopamine receptor numbers, bind-ng characteristics and dopamine uptake (for reviewee 21). Indeed, Lee and co-workers (22) recently re-orted estrogen-induced expression of D5 dopamine re-eptor mRNA in primary cultured neonatal rat hypo-halamic cells. Further, immunoreactive PR wereolocalized in most of the cells expressing D5 dopamineeceptor mRNA after estrogen treatment.In the VMN and ArcN/ME of OVX rats, estrogen

egulates expression of several proteins critical to re-roduction including PR in a sex-specific, time- andose-dependent manner (10). Since the facilitatoryunction of P and D1-like dopamine agonists on lordoticehavior depends on the E-inducible expression of PRn the VMN (2, 23), the present data support the hy-othesis that ovarian steroids and dopamine share aommon convergent pathway in the brain mediated by,t least, the D5 dopamine receptor. In the neural SK--SH cell line (24) and in the medial preoptic area

MPOA) (25), estrogen stimulates intracellular cAMPignaling pathway. Likewise, elevated basal levels ofAMP are detected in 293 cells that stably expressloned human D5 dopamine receptor (26), and as re-orted by Lee (22), in unstimulated CHO cells stablyxpressing the rat D5 dopamine receptor. IntracellularAMP is known to increase gene expression and PRrotein levels by de novo synthesis in some cell types27). More importantly, cAMP/PKA activation mark-dly synergizes with estrogen in ER-mediated tran-criptional activation (for review see 28). Thus, theresence of D5 receptor mRNA in the VMN andrcN/ME after EB-priming raises the possibility that aore active cAMP-dependent protein kinase pathway

xists for fine tuning and facilitation of sex behavior.In summary, we have shown that D5 dopamine re-

eptor mRNA can be detected in the VMN andrcN/ME punchout biopsies of estradiol-primed ovari-ctomized rats. Furthermore, our study also provideshe first direct demonstration of a co-expression of

1/D5 dopamine receptor mRNA in VMN of EB-primedVX rats. Such a co-existence of D5/D1 dopamine re-

eptors has been found in various primate (and rodent)rain regions, such as striatum, cerebral cortex, hip-ocampus, and in the lateral and medial thalamus (29,0). Our data do not allow for any firm conclusionsegarding the cellular and subcellular distributions ofhese co-expressed D5 and D1 dopamine receptorsithin the hypothalamic VMN of the female rat brain.he present study confirms that only D5 dopamineeceptor mRNA is expressed in the ArcN/ME. Furthertudies to determine the cellular and subcellular local-zation of D5 and D1 dopamine receptors with steroideceptors within the VMN and ArcN/ME are underway.

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CKNOWLEDGMENTS

We thank Jennifer E. Lohmann for technical assistance in theanagement of animals and their preparation for experimentation.e are grateful to Child Health Research Center Sequencing Core

ab of Baylor College of Medicine for their technical support. Thisesearch was supported by NICHD/NIH through cooperative agree-ent [U54 (HD07495)] as part of the Specialized Cooperative Cen-

ers Program in Reproduction Research.

EFERENCES

1. Power, R., Mani, S., Codine, J., Conneely, O., and O’Malley,B. W. (1991) Science 254, 1636–1639.

2. Apostolakis, E., Garai, J., Fox, C., Smith, C., Watson, S., Clark,J., and O’Malley, B. (1996) J. Neurosci. 16, 3–15.

3. Missale, C., Nash, S. R., Robinson, S. W., Jaber, M., and Caron,M. (1998) Phsiol. Rev. 78, 189–225.

4. Mani, S., Allen, J., Clark, J., and O’Malley, B. W. (1994) Science265, 1246–1249.

5. Foreman, M., and Moss, R. (1979) Physiol. Behav. 22, 283–289.

6. Mani, S. K., Allen, J. M., Lydon, J. P., Mulac-Jericevic, B.,Blaustein, J. D., DeMayo, F. J., Conneely, O., and O’Malley,B. W. (1996) Mol. Endocrinol. 10, 1728–1737.

7. Strange, P. (1997) in The Dopamine Receptors (Neve, K. A., andNeve, R. L., Eds.), pp. 3–26, Humana Press, Totowa, NJ.

8. Tsai, M. J., and O’Malley, B. W. (1994) Annu. Rev. Biochem. 63,451–486.

9. O’Malley, B. W., Schrader, W. T., Mani, S., Smith, C., Weigel,N. L., Conneely, O. M., and Clark, J. H. (1995) Rec. Prog. Horm.Res. 50, 333–347.

0. Romano, G. J., Mobbs, C. V., Lauber, A., Howells, R. D., andPfaff, D. W. (1990) Brain Res. 536, 63–68.

1. Apostolakis, E. M., Garai, J., Lohmann, J. E., Clark, J. H., andO’Malley, B. W. (1999) Mol. Endocrinol., in press.

2. Parsons, B., Maclusky, N. J., Krey, K., Pfaff, D. W., and McEwen,B. S. (1980) Endocrinol. 107, 774–779.

559

Coordinates, Academic Press Inc., New York.4. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, USA.

5. Vrana, S. L., Kluttz, B. W., and Vrana, K. E. (1995) Brain Res.Mol. Brain Res. 34, 127–134.

6. Yan, Z., Song, W. J., and Surmeier, D. (1997) J. Neurophysiol.77, 1003–1115.

7. Tiberi, M., Jarvie, K. R., Silvia, C., Falardeau, P., Gingrich, J. A.,Godinot, N., Bertrand, L., Yang-Feng, T. L., Fremeau, R. T., andCaron, M. G. (1991) Proc. Natl. Acad. Sci. USA 88, 7491–7495.

8. Sidhu, A. (1998) Mol. Neurobiol. 16, 125–134.9. Sunabara, R. K., Guan, H. C., O’Dowd, B. F., Seeman, P.,

Laurier, L. G., Ng, G., George, S. R., Torchia, J., Van Tol,H. H. M., and Niznik, H. B. (1991) Nature 350, 614–619.

0. Meador-Woodruff, J. H., Mansour, A., Grandy, D. K., Damask,S. P., Civelli, O., and Watson Jr., S. J. (1992) Neurosci. Lett. 145,209–212.

1. Di Paolo, T. (1994) Rev. Neurosci. 5, 27–41.2. Lee, D., Dong, P., Copolov, D., and Lim, A. T. (1999) Mol. Endo-

crinol. 13, 344–352.3. Pfaff, D. W., Schwartz-Giblin, S., McCarthy, M. M., and Kow,

L. M. (1994) in The Physiology of Reproduction (Knobil, E., andNeill, J. D., Eds.), pp. 108–195, Raven Press, Ltd., New York.

4. Watters, J. J., and Dorsa, D. M. (1998) J. Neurosci. 18, 6672–6680.5. Zhou, Y., Watters, J. J., and Dorsa, D. M. (1996) Endocrinol. 137,

2163–2166.6. Tiberi, M., and Caron, M. G. (1994) J. Biol. Chem. 269, 27925–

27931.7. Cho, H., Aronica, S. M., and Katzenellenbogen, B. S. (1994)

Endocrinol. 134, 658–664.8. Katzenellenbogen, B. S. (1996) Biol. Reprod. 54, 287–293.9. Bergson, C., Mrzljak, L., Smiley, J. F., Pappy, M., Levenson, R.,

and Goldman-Rakic, P. S. (1995) J. Neurosci. 15, 7821–7836.0. Choi, W. S., Machida, C. A., and Ronnekleiv, O. K. (1995) Brain

Res. Mol. Brain Res. 31, 86–94.