Ž .Brain Research 800 1998 269–274

Research report

Differential distribution of D dopamine receptors in the brains of several3

mammalian species

Beth Levant )

Department of Pharmacology, Toxicology, and Therapeutics, UniÕersity of Kansas Medical Center, 3901 Rainbow BlÕd., Kansas City, KS 66160-7147,USA

Accepted 12 May 1998

Abstract

The D dopamine receptor has been proposed as a potential target for the treatment of schizophrenia and drug abuse. This study3

compares the distribution of D sites in mouse, rat, guinea pig, and rabbit brain, and dog and human cerebellum using quantitative3w3 xautoradiography with the putatively selective D receptor radioligand H PD 128907. In the mouse, rat, guinea pig, and rabbit, specific3

w3 xH PD 128907 binding was heterogeneously distributed with highest densities observed in the islands of Calleja, followed by the nucleusw3 xaccumbens. Moderate densities of H PD 128907 binding were observed in the anteroventral and dorsomedial caudate nucleus. Dense

w3 xH PD 128907-labelled sites were observed in the dorsal thalamus, posterior mamilliary nucleus, and dorsomedial interpeduncularw3 xnucleus of the rabbit that were not detected in the other species studied. Moderately dense H PD 128907 binding was also observed in

the molecular layer of cerebellar lobule X of the rat but not in the mouse, guinea pig, rabbit, dog, or human. These observations indicatesignificant inter-species differences in the distribution of D receptors. q 1998 Elsevier Science B.V. All rights reserved.3

w3 xKeywords: D dopamine receptor; H PD 128907; Rat; Mouse; Guinea pig; Rabbit; Dog; Human3

1. Introduction

The D dopamine receptor was cloned by Sokoloff et3w xal. in 1990 19 . This receptor, which is similar in sequence

and pharmacology to the D receptor, is of particular2

interest because the receptor and its mRNA appear to beexpressed primarily in brain regions such as the nucleusaccumbens, olfactory tubercle, and islands of Callejaw x4,13,19 , terminal fields of the mesolimbic dopamine pro-jection which has been hypothesized to mediate psychotic

w xsymptoms 20 . Unlike mRNA for the D receptor, very2

low levels of expression of D receptor mRNA are de-3

tected in either the caudate nucleus or the pituitary, brainareas associated with the untoward neurological and en-docrine effects associated with most conventional antipsy-

w xchotics 19 . These observations suggest that the D recep-3

tor, alone or in conjunction with other receptors, may be atarget for novel antipsychotic drugs which might be free of

) Fax: q1-913-588-7501; E-mail: blevant@kumc.edu

extrapyramidal effects. A role for the receptor in thereinforcing properties of cocaine has also been proposedw x5 .

Thus far, the distribution of D receptor mRNA and D3 3

receptors have been extensively mapped only in rat brainw x2,4 . Consistent with initial reports, these studies indicatethe preferentially limbic localization of D receptors in rat3

brain. These studies have also revealed the somewhatw xsurprising presence of D sites in rat cerebellum 4,12 .3

High densities of D sites are also observed in the nucleus3w xaccumbens and islands of Calleja of human brain 8,15 ;

however, many brain areas have yet to be examined.Clearly, one of the most striking features of the D3

receptor is its relatively restricted distribution in brainwhich has lead to numerous hypotheses regarding thereceptor’s function and potential therapeutic significance.Accordingly, the purpose of this study was to compare thedistribution of D sites in the brains of several mammalian3

species. This study will show that while the distribution ofD receptors is generally similar in the basal ganglia and3

limbic forebrain of the species studied, notable differencesare observed in hypothalamic, thalamic, mesencephalic,and cerebellar brain areas.

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 98 00529-0

( )B. LeÕantrBrain Research 800 1998 269–274270

2. Materials and methods

2.1. Tissues

All experiments were carried out in accordance with theDeclaration of Helsinki and the NIH Guide for the Careand Use of Laboratory Animals. Adult, male Sprague–

Ž .Dawley rats 175–200 g; Harlan Labs, Indianapolis, INŽ .and 129-OLA mice Jackson Labs, Bar Harbor, ME were

housed with free access to laboratory chow and water. Thetemperature- and humidity-controlled animal facility had a12 h dark–light cycle. Animals were obtained at least 5days before the commencement of each experiment andwere accustomed to handling. Rats and mice were sacri-ficed by decapitation, the brains rapidly removed andfrozen in isopentane.

Guinea pig, rabbit, and dog brains were purchased fromŽ .Pel-Freez Rogers, AZ . Human cerebellar lobule X from

Žnormal subjects 3 male, 1 female; age 59–77 years;.post-mortem delay 70–169 min was generously donated

by the Kathleen Price Bryan Brain Bank, Duke UniversityŽ .Medical Center Durham, NC . All brains were stored at

y708C.

[3 ]2.2. H PD 128907 autoradiography

w3 xH PD 128907 autoradiography was performed as pre-w x Ž .viously described 2 . Brain sections 20 mm were cut on

a cryostat, thaw-mounted onto chrome-alumrgelatin-coated slides and stored at y708C until use. Slide-mountedbrain sections were then brought to room temperature andallowed to dry thoroughly. Duplicate sections from thesame animalrsubject were used for each data point. Slides

w3 xwere incubated with ;0.7 nM H PD 128097 in assayŽ .buffer 50 mM Tris, 1 mM EDTA, pH 7.4 at 238C for 2 h

at 238C. Nonspecific binding was defined in the presenceof 1 mM spiperone. Following incubation, slides weredipped in ice-cold assay buffer, washed for two consecu-tive 2 min periods in ice-cold assay buffer, dipped inice-cold deionized H O, and dried under a cool air stream.2

Radiolabeled sections were subsequently apposed to 3H-Ž .Hyperfilm Amersham, Arlington Heights, IL with 20 mm

w3 xH methylmethacrylate autoradiographic standardsŽ . 3Amersham for a period of 12–14 weeks. H-Hyperfilmwas developed according to the manufacturer’s instruc-tions. Brain sections were then stained with Cresyl violet.

Autoradiographic images were digitized and quantifiedusing the Macintosh-based video densitometry programNIH ‘Image’ version 1.4. Best-fit curves of optical density

w3 xgenerated by the H methylmethacrylate autoradiographicstandards resulted when a Rodbard plot was used to de-scribe the relationship between optical density and radioac-tivity. Brain regions were identified according to various

w xbrain atlases 14,16,18,21,22 . Autoradiograms of coronal

Table 1Distribution of D dopamine receptors in the fore- and midbrain of several mammalian species3

3w x Ž .Brain regionrspecies H PD 128907 binding fmolrmg tissue equivalent

Mouse Rat Guinea pig Rabbit

Frontal cortex 0.06"0.06 0.63"0.4 0.45" .041 0.26"0.22Nucleus accumbens 24"6.1 20"3.6 7.2"0.8 8.1"0.3CaudateAnteroventral 29"9.2 6.6"1.2 5.1"0.4 4.1"1.2Dorsomedial 10"1.0 4.9"0.4 4.7"0.7 2.4"0.3Central 2.3"0.4 0.49"0.28 2.9"0.1 0.9"0.5

Islands of Calleja 53"11 50"16 21"2.0 26"3.1Globus pallidus 6.3"0.9 0.95"0.34 2.4"0.03 0.26"0.15Cingulate cortex 2.8"2.8 0.42"0.17 0.53"0.15 0.43"0.43Hypothalamus 5.2"1.6 1.7"0.79 1.2"0.1 0.20"0.16Dorsal premammilary nucleus 3.3"0.4

Thalamus 0.6"0.32 1.6"0.79 0.58"0.28Lateral dorsal nucleus 5.5"1.1Mediodorsal nucleus 2.0"0.2Interomediodorsal nucleus 6.2"1.0

Hippocampus N.D. 0.91"0.41 0.64"0.21 1.2"1.2Amygdala N.D. 2.9"1.9 0.53"0.15 N.D.Substantia nigra 1.29"1.29 4.4"0.18 0.09"0.09 0.95"0.36Interpeduncular nucleusDorsomedial N.D. N.D. N.D. 3.8"1.7

Data were derived from quantification of autoradiograms produced from 3–4 animals and are expressed as the mean"S.E.M. Coronal sections werew3 xincubated with ;0.7 nM H PD 128907.

Nonspecific binding was defined in the presence of 1 mM spiperone.Ž . w3 xThe unit of receptor density fmolrmg tissue equivalent is that supplied by the manufacturer of H methylmethacrylate autoradiography standards

Ž .Amersham .The results have not been corrected for differential quenching of tritium by gray and white matter.N.D.—not detected.

( )B. LeÕantrBrain Research 800 1998 269–274 271

w3 x Ž . Ž . Ž . Ž . Ž .Fig. 1. Autoradiographic distribution of H PD 128907-labeled sites in striatum. A mouse. B rat. C rat, nonspecific binding. D guinea pig. Erabbit. Data shown represent total binding unless otherwise noted. Specific autoradiograms are representative of those obtained from each of four animals.

Ž .The darker areas of the autoradiograms indicate regions of higher receptor density. Slide-mounted coronal brain sections 20 mm were incubated withw3 x;0.7 nM H PD 128907 as described in Section 2. Nonspecific binding was defined by 1 mm spiperone. Quantified data from these autoradiograms are

summarized in Table 1. Abbreviations used: Acb—nucleus accumbens, dm—dorsomedial caudate, ICj—islands of Calleja. Bars5 mm.

sections were sampled bilaterally. Measurements representaverage pixel optical density by volume analysis. Results,expressed as fmolrmg tissue equivalent, were not cor-rected for differential quenching by white and gray matter.

3. Results

w3 xH PD 128907-labeled sites were heterogeneously dis-tributed in the brains of mouse, rat, guinea, and rabbit. The

w3 xoverall density of H PD 128907 binding was similar inw3 xthe mouse and rat. The overall density of H PD 128907

binding was also similar in the guinea pig and rabbit butw3 xthese species exhibited roughly half the density of H PD

128907-labeled sites observed in the mouse and rat.w3 xIn all species studied, H PD 128907 binding was

observed in highest densities in the islands of Calleja,Ž .followed by the nucleus accumbens Table 1, Fig. 1 .

w3 xModerate densities of H PD 128907 binding were ob-served in the anteroventral and dorsomedial portions of the

w3 x Ž . Ž . Ž . Ž . Ž .Fig. 2. Autoradiographic distribution of H PD 128907-labeled sites in thalamus. A mouse. B rat. C guinea pig. D rabbit. E rabbit, nonspecificbinding. Autoradiograms were generated as described for Fig. 1. Quantified data from these autoradiograms are summarized in Table 1. Abbreviationsused: IMD—interomediodorsal nucleus, MD—mediodorsal nucleus, LD—lateral dorsal nucleus. Bars5 mm.

( )B. LeÕantrBrain Research 800 1998 269–274272

w3 x Ž . Ž . Ž . Ž . Ž .Fig. 3. Autoradiographic distribution of H PD 128907-labeled sites in hypothalamus. A mouse. B rat. C guinea pig. D rabbit. E rabbit,nonspecific binding. Autoradiograms were generated as described for Fig. 1. Quantified data from these autoradiograms are summarized in Table 1.Abbreviations used: PMD—dorsal premammillary nucleus. Bars5 mm.

caudate nucleus with relatively little binding in the centralw3 xportion of the nucleus. Relatively little H PD 128907

binding was detected in the globus pallidus.In the mouse, rat, and guinea pig relatively low densi-

w3 xties of H PD 128907 were detected in the thalamus andw3 xhypothalamus. In the rabbit, however, H PD 128907

Ž .binding was detected in the dorsal thalamic nuclei Fig. 2 .w3 xRelatively dense H PD 128907 binding was observed in

the interomediodorsal and lateral dorsal nuclei and moder-ately dense binding in the mediodorsal nucleus. In the

w3 xrabbit hypothalamus, moderately dense H PD 128907binding was detected in the posterior mammillary nucleusŽ .Fig. 3 .

w3 xH PD 128907-labeled sites in the substantia nigraŽ .were of highest density in the rat Fig. 4 . Lower, but

detectable labelling of the substantia nigra was also ob-

w3 x Ž . Ž . Ž . Ž . Ž .Fig. 4. Autoradiographic distribution of H PD 128907-labeled sites in the mesencephalon. A mouse. B rat. C guinea pig. D rabbit. E rabbit,nonspecific binding. Autoradiograms were generated as described for Fig. 1. Quantified data from these autoradiograms are summarized in Table 1.Abbreviations used: SN—substantia nigra, IPDM—dorsomedial interpeduncular nucleus. Bars5 mm.

( )B. LeÕantrBrain Research 800 1998 269–274 273

Table 2Distribution of D dopamine receptors in the cerebellum of several3

mammalian species3w x Ž .Speciesr H PD 128907 binding fmolrmg tissue equivalent

region Lateral lobe Vermis Lobule XaRat 0.10"0.086 N.D. 9.9"2.0

Mouse N.D. 0.58"0.58 1.0"1.0Guinea pig 0.60"0.50 1.15"1.04 0.09"0.09Rabbit 0.04"0.04 0.26"0.22 0.40"0.36Dog N.D. 0.09"0.09 0.20"0.15Human – – 0.49"0.23

Data were derived from quantification of autoradiograms produced from3–4 animalsrsubjects and are expressed as the mean"S.E.M.

w3 xSagittal sections were incubated with ;0.7 nM H PD 128907.Nonspecific binding was defined in the presence of 1 mM spiperone.

Ž .The unit of receptor density fmolrmg tissue equivalent is that suppliedw3 xby the manufacturer of H methylmethacrylate autoradiography stan-

Ž .dards Amersham .The results have not been corrected for differential quenching of tritiumby gray and white matter.N.D.—not detected.a Molecular layer.

served in the mouse and rabbit. Very low density ofw3 xH PD 128907-labeled sites, if any, were detected in thesubstantia nigra of the guinea pig.

w3 xVery low levels of H PD 128907 binding were ob-served in the hippocampus of all species studied. Likewise,

w3 xnegligible H PD 128907 binding was detected in othermidbrain structures of the mouse, rat, and guinea pig. In

w3 xthe rabbit however, moderately dense H PD 128907binding was observed in the dorsomedial interpeduncular

Ž .nucleus Fig. 4 .w3 xModerately dense H PD 128907 binding was observed

discretely in the molecular layer of cerebellar lobule X ofŽ . w3 xthe rat Table 2, Fig. 5 . Very low densities of H PD

128907-labelling were observed in the lateral lobe, vermis,or lobule X of the mouse, guinea pig, or rabbit. Likewise,

w3 xvery low densities of H PD 128907 binding were ob-served in lobule X of the dog and the human.

4. Discussion

w3 xThis study used the radioligand H PD 128907 todetermine the localization of D dopamine receptors in the3

brains of various mammalian species. This radioligand hasbeen shown to exhibit significant selectivity for a subset ofdopaminergic sites with a pharmacological profile, distri-bution, and lack of guanyl nucleotide regulation appropri-

w xate for the D receptor 1,2,8 .3

The three rodent species and the lagomorph exhibitedsimilar distributions of D sites in the basal ganglia and3

limbic forebrain similar to that previously reported in theŽ w x.rat and human for review, see Ref. 11 . This is concor-

dant with previous studies that indicate that the distribu-tions of D - and D -like receptors are generally similar in1 2

the basal ganglia of mammalian and non-mammalianw xspecies 6,17 . This distribution is also consistent with the

proposed role of the receptor in locomotor behavior andŽ w x.reinforcement and reward for review, see Ref. 11 .

Notable differences in the distribution of D sites,3

however, were observed in limbic hypothalamic, thalamic,and mesencephalic brain areas. Specifically, the rabbitexhibited moderately dense to dense expression of D sites3

in the dorsal thalamus, posterior mammillary nucleus anddorsomedial interpeduncular nucleus that was not observedin the rodent species. This suggests that in the rabbit, D3

receptors may play a more extensive role in limbic func-tions than in rodent species.

Another notable inter-species difference is the detectionof moderately dense D binding of the molecular layer of3

cerebellar lobule X of the rat. Expression of dopamine

w3 x Ž . Ž . Ž . Ž . Ž .Fig. 5. Autoradiographic distribution of H PD 128907-labeled sites in cerebellum. A mouse. B rat. C rat, nonspecific binding. D guinea pig. EŽ . Ž .rabbit. F dog. G human, lobule X. Autoradiograms were generated using sagittal brain sections of medial cerebellum as described for Fig. 1. Quantified

data from these autoradiograms are summarized in Table 2. Arrow indicates lobule X. Bars5 mm.

( )B. LeÕantrBrain Research 800 1998 269–274274

receptors in this brain area was not observed in the otherrodent species studied nor in the rabbit, dog, or human.

w xThis observation concurs with that of Camps et al. 6indicating the presence of D -like dopamine receptors in2

cerebellar lobule X of the rat but not the mouse, guineapig, or cat. The detection of D sites in lobule X, or3

vestibulocerebellum, of the rat suggests a possible role inw xthe control of posture, muscle tone, or eye movements 7 .

The functional role of these cerebellar D receptor has not3

been thoroughly explored; however, microinjection of adopamine antagonist into the rat vestibulocerebellum was

w xreported to produce alterations in locomotor activity 3 .Based on initial data on the regional distribution of D3

sites, hypotheses have been formulated about the potentialutility of the site in the treatment of human disease. Thusfar, the distribution of the D receptor in human brain has3

been studied in only a limited number of brain regions.Highest densities of D sites are reported in the nucleus3

w xaccumbens and islands of Calleja 8,15 . Moderate amountsof D binding were observed in the basal ganglia, parietal,3

temporal and occipital cortex, and cerebellar cortex, fol-lowed by substantia nigra, hippocampus, and the basolat-

w xeral, lateral and basomedial amygdaloid nuclei 9,10,15 .D receptors were also detected in moderate density in the3

pituitary, with somewhat greater labelling in the posteriorw xlobe than the anterior 9 . Although some thalamic areas

w xhave been examined 9 , the distribution of D sites in3

hypothalamus and thalamus have yet to be scrutinized indetail. The present observations indicate significant inter-species in the distribution of D receptors. As such, hy-3

potheses based on the distribution of sites in a particularspecies may not generalize to other species. Likewise, thepharmacological effects of drugs acting at the D site may3

vary significantly between species. Thus, caution must beexercised in selecting animal models for the study ofD -mediated effects. Moreover, with the current interest in3

the D receptor as a therapeutic target, these findings3

underscore the importance of studying the distribution ofthese sites in human brain in detail.

Acknowledgements

The author thanks the Kathleen Price Bryan BrainBank, Duke University Medical Center, Durham, NC forthe donation of human cerebellar tissues, Kimberly Mor-gan for technical assistance, and Dr. Nancy Berman foranatomical consultation. Supported by NIMH MH 52839.

References

w x1 H.C. Akunne, P. Towers, G.J. Ellis, D. Dijkstra, H. Wikstrom, T.G.Heffner, L.D. Wise, T.A. Pugsley, Characterization of binding ofw3 xH PD 128907, a selective dopamine D receptor agonist ligand, to3

Ž .CHO-K1 cells, Life Sci. 57 1995 1401–1410.

w x2 G.N. Bancroft, K.A. Morgan, R.J. Flietstra, B. Levant, Binding ofw3 xH PD 128907, a putavively selective ligand for the D dopamine3

receptor, in rat brain: a receptor binding and quantitative autoradio-Ž .graphic study, Neuropsychopharmacology 18 1998 305–316.

w x3 S. Barik, R. Debeaurepaire, Evidence for a functional role of theŽ .dopamine D receptors in the cerebellum, Brain Res. 737 19963

347–350.w x4 M.L. Bouthenet, E. Souil, M.P. Martres, P. Sokoloff, B. Giros, J.C.

Schwartz, Localization of dopamine D receptor mRNA in the rat3

brain using in situ hybridization histochemistry: comparison withŽ .dopamine D receptor mRNA, Brain Res. 564 1991 203–219.2

w x5 S.B. Caine, G.F. Koob, Modulation of cocaine self-administration inŽ .the rat through D-3 dopamine receptors, Science 260 1993 1814–

1816.w x6 M. Camps, P.H. Kelly, J.M. Palacios, Autoradiographic localization

of D and D receptors in the brain of several mammalian species, J.1 2Ž .Neural Transm. 80 1990 105–127.

w x7 C. Ghez, S. Fahn, The cerebellum. In: E.R. Kandel, J.H. SchwartzŽ .Eds. , Principles of Neural Science, ElsevierrNorth-Holland, NewYork, 1981, pp. 334–346.

w x8 H. Hall, C. Halldin, D. Dijkstra, H. Wikstrom, L.D. Wise, T.A.Pugsley, P. Sokoloff, S. Pauli, L. Farde, G. Sedvall, Autoradio-graphic localization of D -dopamine receptor in the human brain3

Ž . w3 xusing the selective D -dopamine receptor agonist q - H PD3Ž .128907, Psychopharmacology 128 1996 240–247.

w x9 L. Herroelen, J.-P. De Backer, N. Wilczak, A. Flamez, G. Vaquelin,J. De Keyser, Autoradiographic distribution of D -type dopamine3

w3 xreceptors in human brain using H 7-hydroxy-N, N-di-n-propyl-2-Ž .aminotetralin, Brain Res. 648 1994 222–228.

w x10 R.A. Lahti, R.C. Roberts, C.A. Tamminga, D -family receptor dis-2

tribution in human post-mortem tissue: an autoradiographic study,Ž .NeuroReport 6 1995 2505–2512.

w x11 B. Levant, The D dopamine receptor: neurobiology and potential3Ž .clinical relevance, Pharmacol. Rev. 49 1997 231–252.

w x12 B. Levant, E.B. DeSouza, Differential pharmacological profile ofw3 xstriatal and cerebellar dopamine receptors labeled by H quinpirole:

identification of a discrete population of D receptors, Synapse 143Ž .1993 90–95.

w x13 B. Levant, D.E. Grigoriadis, E.B. DeSouza, Characterization ofw3 xH quinpirole binding to D -like dopamine receptors in rat brain, J.2

Ž .Pharmacol. Exp. Ther. 262 1992 929–935.w x14 T.J. Luparello, Stereotaxic Atlas of the Forebrain of the Guinea Pig,

The Williams & Wilkins, Baltimore, 1967.w x15 A.M. Murray, H.L. Ryoo, E. Gurevich, J.N. Joyce, Localization of

dopamine D receptors to mesolimbic and D receptors to mesostri-3 2

atal regions of human forebrain, Proc. Natl. Acad. Sci. USA 91Ž .1994 11271–11275.

w x16 G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates,2nd edn., Academic Press, Sydney, 1986.

w x17 E.K. Richfield, A.B. Young, J.B. Penney, Comparative distributionof dopamine D-1 and D-2 receptors in the basal ganglia of turtles,

Ž .rats, cats, and monkeys, J. Comp. Neurol. 262 1987 446–463.w x18 B.M. Slotnik, C.M. Leonard, A Stereotaxic Atlas of the Albino

Mouse Forebrain, U.S. Dept. of Health Education and Welfare,Rockville, 1975.

w x19 P. Sokoloff, B. Giros, M.P. Martres, M.L. Bouthenet, J.C. Schwartz,Molecular cloning and characterization of a novel dopamine receptorŽ . Ž .D as a target for neuroleptics, Nature 347 1990 146–151.3

w x20 J.R. Stevens, An anatomy of schizophrenia?, Arch. Gen. PsychiatryŽ .29 1973 177–189.

w x21 L.W. Swanson, Brain Maps: Structure of the Rat Brain, Elsevier,Amsterdam, 1992.

w x22 D. Winkler, A. Potter, An Anatomical Guide to Experimental Re-searches on the Rabbit’s Brain, A.L. Versluys, Amsterdam, 1911.