Proc. Nati. Acad. Sci. USAVol. 89, pp. 11988-11992, December 1992Neurobiology

Immunohistochemical localization of the D1 dopamine receptor inrat brain reveals its axonal transport, pre- and postsynapticlocalization, and prevalence in the basal ganglia, limbicsystem, and thalamic reticular nucleus

(cortex/hippocampal formation/hypotaamus/caudake-.utamen/substantla I)

QIN HUANG*t, DAN ZHOU*t, KATHY CHASES, JAMES F. GUSELLA§¶, NEIL ARONINt,AND MARIAN DIFIGLIA*t II*Laboratory of Cellular Neurobiology, WMolecular Neurogenetics Unit, and Departments of tNeurology and lGenetics, Massachusetts General Hospital andHarvard Medical School, Boston, MA 02114; and tDepartments of Medicine and Cell Biology, University of Massachusetts Medical School, Worcester,MA 01655

Communicated by Patricia S. Goldman-Rakic, August 20, 1992

ABSTRACT DI d .pine receptor llization was exam-ined by i unohistochemistry using a polyclonal anti-peptideantibody which (i) immunoprecipitated a protein frmentencoded by a D1 receptor cDNA and (it) on Western blots ofsolubilized striatal and hippocampal membranes recognizedtwo proteins ofapproimatey 50 kDa and 75 kDa, correspond-ing to reported sizes ofDI receptor proteins. Immunreactivoverlapped with dopamine-containing pathways, patterns ofD1receptor binding, and mRNA expression. Staning was con-centrated in prefrontal, cigulate, parietal, piriform, entorhi-nal, and hippocampal cortical areas and subcorticafly in thebasal ganglia, amygdala, septal area, substanti inomnta,thalamus, hypothalamus, and neurohypophysis. Prominentlabeling was seen in the thalic reticular nucleus, a regionknown to integrate ascending basal forebrain inputs withthalamocortical and corticothalamic pathways and in fiberbundles interconnecting limbic areas. In striatal neuropil,staining appeared in spines (heads and necks), at postsynapticsites in dendrites, and in axon terminals; in the pars reticulataof the substantia nigra, labeling was prevalent in myelinatedand unmyelinated axons and dendrites. These data providedirect evidence for the regional and subcellular distribution ofD1 receptor protein in the brain and for its pre- and postsyn-aptic localization in the basal ganglia. The prominent immu-noreactivity seen in the limbic system and thalamic reticularnucleus supports an important role for this receptor subtype inmediating integrative processes involved with learning, mem-ory, and cognition.

Two classes of dopamine receptors, D1 and D2, are known tomediate the diverse functional effects of dopamine neuro-transmission in the brain. These receptor classes are coupledto guanine nucleotide-binding proteins and have distinctproperties based on pharmacological, biochemical, and elec-trophysiological data (1, 2). D1 receptors are more abundantin brain than D2 receptors, but the precise role ofD1 receptoractivation independent of D2 receptor actions has not beenestablished (3). D1 receptor stimulation may modulate theactivity of D2 receptors (4), mediate stereotypic behaviors(5), alter the release of neurotransmitters (6), affect electro-encephalograph activity and behavioral arousal (7), and af-fect memory processes associated with the prefrontal cortex(8). D1 receptor activity shows age-dependent decreases inthe human brain (9) and is altered in Huntington disease (10)and Alzheimer disease (11).

Receptor binding shown by autoradiography and mRNAlocalization with in situ hybridization show that D1 receptorbinding activity and expression are highest in the basalganglia system (12-15), but also widespread in brain, al-though reports vary as to the extent. Although biochemicaland pharmacological studies have suggested functional cir-cuits in which D1 receptors may be localized presynapticallyas well as postsynaptically, the above methods cannot re-solve the location and abundance of receptor protein withinindividual neurons. Moreover, there is in some brain areas apoor correspondence between D1 receptor binding andmRNA expression, suggesting transport of the receptor pro-tein (15). This interpretation requires examination of thesubcellular compartments where the receptor protein re-sides. Establishing the occurrence ofD1 receptors at synapticand nonsynaptic sites in plasma membranes can help deter-mine whether dopamine exerts a neurotransmitter or hor-mone-like effect.To obtain more precise cellular information about D1

receptors, we examined their localization by imunohis-tochemistry. Peptide immunogens have been used success-fully to generate antibodies which specifically recognize D2receptors (16). In this study we developed a polyclonalantibody directed to a peptide sequence located in theC-terminal cytoplasmic domain of the D1 receptor molecule.

METHODSAntibody Preparation and Purification. The anti-Di recep-

tor antiserum was produced in a New Zealand White rabbit(Assay Research, College Park, MD) against a 13-residuepeptide corresponding to amino acids 403-415 predicted forthe cloned D1 receptor (13, 17, 18). The peptide sequenceshowed no similarity with other known sequences of cate-cholamine receptors or with other dopamine receptor sub-types (D2, D3, D4, and D5) (19-22). One milligram of thepeptide was conjugated with 150 ,ug of keyhole limpet hemo-cyanin, after addition of 0.5% glutaraldehyde (overnight,room temperature). An immunogenic enhancer was added tothe mixture, which was emulsified with an equal volume ofFreund's complete adjuvant and injected at a single site intothe rabbit, followed 2 weeks later by a second injection andthen by three booster immunizations at 1-month intervals.Sera from blood samples were collected bimonthly and storeduntil further analysis. A solid-phase ELISA with immobilizedsynthetic peptide was used to monitor antibody production.

ItTo whom reprint requests should be addressed at: Laboratory ofCellular Neurobiology, Massachusetts General Hospital, MGH-east, Charlestown, MA 02129.

11988

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Antisera were applied to a protein G-Sepharose (Sigma)column (23) or a 6-aminohexanoic acid N-hydroxysuccini-mide ester-Sepharose 4B (Sigma) column to which the pep-tide immunogen was bound (23). Elution was accomplishedby the addition of 100 mM glycine, pH 3.5, into Tris buffer,pH 8.0. The antiserum was dialyzed against phosphate-buffered saline (PBS, pH 7.4), overnight at 40C, and stored at-700C.Preparation of Membrane Extracts. The protocol of Kelle-

her et al. (24) was used with the following modifications: useof brain tissue, addition of dithiothreitol (0.5 mM) andmultiple protease inhibitors (phenylmethanesulfonyl fluo-ride, 0.5 mM; pepstatin, 1 pug/ml; L-1-tosylamido-2-phenylethyl chloromethyl ketone, 70 Ag/ml; leupeptin, 0.5gg/ml; and soybean trypsin inhibitor, 10 gg/ml), and addi-tion of SKF 38393 (Research Biochemicals, Natick, MA) to30 ,uM after each pellet resuspension. The solubilized mem-branes were stored at 40C and used within 1 day.

Immunoprecipitation Analysis. A cDNA (1050 bases,pBluescript II KS, gift of Stephen Fink) (MassachusettsGeneral Hospital, Boston) encoding a 38-kDafragment oftherat D1 receptor, including amino acids 403-415, was tran-scribed to produce mRNA (Stratagene kit), which was thenused in reticulocyte lysates containing [35S]methionine toproduce a radiolabeled D1 receptor protein of 38 kDa, asverified by SDS/10% PAGE. Affinity-purified anti-peptideantibodies (final concentration, 1.8 ,ug ofprotein per ml) wereincubated with 40 ,ug of the radiolabeled D1 receptor proteinin a volume of 500 ,ul at 4°C overnight. Protein A-Sepharose(20 ,u of wet beads) was added (1 hr, room temperature) andthe mixture was centrifuged. The pellet was subjected toSDS/12% PAGE, and the immunoprecipitated protein(s) wasdetected by autoradiography of the dried gel (23).Western Blot Analysis. Solubilized membranes from neo-

striatum and hippocampus (three rats for each set ofextracts)were submitted to SDS/10o PAGE. The proteins wereblotted to Immobilon-P (Millipore). The blots were treatedwith affinity-purified anti-DI receptor antibodies (1.8 gg/ml),preimmune antibodies prepared by passage over the peptideaffinity column, and affinity-purified anti-D1 receptor anti-bodies that were preincubated with the D1 receptor peptide(100 ,ug/ml). The blots were then treated with alkalinephosphatase-conjugated goat anti-rabbit antiserum, followedby nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.

Animals, Tissue Preparation, and Immunohistochemistry.Sprague-Dawley rats (250-350 g) were anesthetized withxylazine and ketamine (0.1 and 0.5 mg/kg) and perfusedthrough the heart with 400 ml of4% paraformaldehyde (plus0.1% glutaraldehyde, in some cases) in 0.1 M phosphatebuffer. Coronal or sagittal sections, 40 ,um thick, were cut ona Vibratome, transferred to 2-10%o normal goat serum in PBSfor 1 hr, washed in PBS, and incubated for 24-36 hr in PBScontaining the anti-D1 receptor antibody at various proteinconcentrations (0.7-2.9 ,ug/ml for protein G-purified antise-rum and 20 ,ug/ml for affinity-purified antibody). Sectionswere processed with the biotin-avidin immunoperoxidasemethod (Vectastain kit; Vector Laboratories) and preparedfor light and electron microscopy (25). Controls includedomission of the primary antiserum, use of preimmune serum(1:500 to 1:2000), and preabsorption of the anti-DI antiserumwith excess peptide (1.25 mg/ml) prior to tissue incubation.Under these control conditions, staining was absent in brain.In addition, the anti-D1 receptor antibody failed to stain ratliver, a tissue devoid of D1 receptors.

RESULTSCharacterization of the DI Receptor Antiserum. In the

ELISA, antiserum (1:104 to 1:106) bound specifically to

immobilized peptide. Preimmune serum binding was negli-gible in the same concentrations (data not shown). To showinteractions of the antiserum with the DI receptor itself, an35S-labeled D1 receptor fragment of 38 kDa was synthesized(see Methods), immunoprecipitated with affinity-purified an-tiserum, and separated by SDS/PAGE. Autoradiographyrevealed an immunoprecipitated protein of the expectedmolecular mass (Fig. 1A). Western blot analysis documentedthat the affinity-purified antiserum recognized native pro-teins in the neostriatum and hippocampus (Fig. 1B) at 50 and75 kDa. Preimmune antiserum lacked crossreactivity withnative brain proteins (Fig. 1B), as did affinity-purified anti-serum preincubated with D1 receptor peptide (data notshown).D 1b of the DI Recptr. Immunoreactive neurons,

seen in rat forebrain, midbrain, and hindbrain, were charac-terized by moderate to heavy deposits of punctate reactionproduct along plasma membranes and/or within cytoplasm.Protein G-purified serum produced cell body and neuropilstaining in many regions, including the basal ganglia, hippo-campus, and cortex. Affinity purification of the serum sig-nificantly enhanced the staining ofneuropil (dendrites, fibers,and axon terminals) in all areas and cell groups and processesin parts of the thalamus, hypothalamus, and midbrain. Therat atlas of Paxinos and Watson (26) was used as a referencefor the brain regions identified below.

Cortex. Immunoreactive neurons were seen primarily inprefrontal, lateral orbital, frontal, parietal (Fig. 2 A and B),and cingulate (Fig. 2 C and D) cortices, especially withinlayers II/III and V/VI (Fig. 2 A and C), and in the piriformand entorhinal cortices, where they were concentrated inlayer II. Labeled neurons had features of pyramidal cells(Fig. 2 B and D) and exhibited dense punctate staining alongplasma membranes. Affinity purification of the antiserumreduced the labeling of plasma membranes but markedlyenhanced the staining of neuropil in layers I and VI and oflabtled neurons in the pericallosal region of layer VI.Hippocampal formation. The plasma membranes of py-

ramidal cells (Fig. 2 Eand F) in all CA fields, especially CA2,were heavily stained along with the neuropil of the stratumoriens and stratum radiatum. In the dentate gyrus (Fig. 2 Eand G), immunoreactivity appeared in the cytoplasm ofgranule-cell bodies and in dendrites in the molecular layer(Fig. 2G).Basal ganglia and other subcortical forebrain nuclei.

Prominent staining of neurons and neuropil was presentthroughout the caudate-putamen and nucleus accumbens

AkDa97-

46-

30-

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69 -

46 -

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'A-

21-

i1 2 3 4

FIG. 1. Characterization of anti-D1 receptor antiserum. (A) Im-munoprecipitation of 35-labeled D1 receptor protein (arrow) syn-thesized in vitro using D1 receptor protein cDNA. (B) Western blotanalysis of solubilized membranes prepared from neostriatum (lanes1 and 3; 200 pg of protein) and hippocampus (lanes 2 and 4; 400 pgof protein). Coomassie staining of the gel after transfer revealed that.80%o of protein was transferred to the blot. Preimmune serum wasused for lanes 1 and 2; anti-Di receptor affinity-purified antiserumwas used for lanes 3 and 4. Arrows indicate protein bands of 50 and75 kDa. Molecular mass standards are shown at left in A and B.

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CAii ;Aj

and included numerous medium-sized striatal neurons (Fig.3A). Of 1718 striatal neurons identified with Nomarski inter-ference microscopy, 49% had D1 receptor immunoreactivity.At the electron microscopic level, reaction product in thestriatum was seen in the heads (Fig. 3G) and necks (Fig. 31)of spines, in dendritic shafts at postsynaptic sites apposed tosymmetric synapses (Fig. 3H), and in axon terminals (Fig.4B), which formed asymmetric synapses with dendriticspines. The neuropil of the globus pallidus and the islands ofCalleja were D1 receptor-positive. Staining was high in neu-rons of the ventral pallidum, the substantia inominata, theamygdala (medial division of the central nucleus and thecortical amygdala), the ventral endopiriform nucleus, andparticularly the olfactory tubercle. Cell-body and neuropilstaining appeared throughout the septal area and was highestin the dorsal division of the lateral septal nucleus.

Thalamus. Intense neuronal labeling of cell bodies, den-drites, and fibers was seen in the reticular nucleus and itsextension with the zona incerta (Fig. 3B). Since no priorreports have been made of dopamine innervation to thereticular nucleus, we immunostained sections for tyrosinehydroxylase and found that positive axons traverse thereticular nucleus where they form symmetric axodendriticsynapses (Fig. 4A). Neuropil staining was high in the lateralgeniculate nucleus and the lateral dorsal thalamic nucleus(Fig. 3C). Labeled neurons were numerous in the medialdorsal nucleus, where immunoreactivity was confined to thecytoplasm of somata and proximal processes, some of whichlooked like axon initial segments (Fig. 2H).

X A . FIG. 2. DI receptor immuno-* r idreactivity in parietal cortex (A and

B) cingulate cortex (C and D),hippocampal formation (E-G,and medial dorsal nucleus of thethalamus (H). In A and C, num-bers identify cortical layers, andboxed areas are shown inB andDrespectively. Arrow in Gidentifiesllabeled dentate granule cell. [Bars

_Lg = 100 Am (A, C, and E) or 50pm(B. D, and F-H).]

Midbrain. Prominent staining of neuropil and some cellbodies was found in the superficial gray layer of the superiorcolliculus. The neuropil of the central gray, the oculomotorcomplex, and the ventral tegmental area was immunoreactive.In the substantia nigra pars lateralis, neurons displayed intensestaining along plasma membranes. The pars reticulata con-tained immunoreactive dendrites and a dense concentration oflabeled fibers and axon terminals, particularly in the regionadjacent to the cerebral peduncle. Ultrastructural study re-vealed reaction product within myelinated and unmyelinatedaxons (Fig. 31), vesicle-containing terminals, and dendrites.Hypothalamus and pituitary. Intense staining was seen in

axon terminals and somata of the periventricular and arcuatenuclei, in fibers of the inner lamina of the median eminence,and within a dense plexus of axons and terminals in themedial and lateral regions of the hypothalamus and theparaventricular and supraoptic nuclei. Punctate neuropillabeling was high in the lateral mammlla and the supra-mmillary nuclei, moderate in the medial mammillary nu-cleus (lateral), and low in the medial mammillary nucleus(Fig. 3F). Staining was seen in the suprachiasmatic nucleus.In the pituitary (Fig. 3D), labeling appeared intense in theneural lobe, light in the anterior lobe, and absent from theintermediate lobe.

Cerebellum. Immunoreactivity was prominently localizedto Golgi cells (Fig. 3E) in the granule-cell layer, to somePurkinje cells, and to the neuropil of the molecular layer.Fiber bundles. Immunoreactivity was concentrated in fiber

bundles which connect limbic forebrain nuclei, including the

11990 Neurobiology: Huang et al.

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FIG. 3. D1 receptor immunoreactiv-ity in the caudate-putamen (A); the re-ticular nucleus (Rt) and zona incerta (ZI)(B); laterodorsal thalamic nucleus, dor-somedial part (LDDM) and stria termi-nalis (st) (C); neural lobe (NL) and ante-rior lobe (AL) (D); cerebellar cortex (E);mammillary complex (F); and neuropil ofthe caudate-putamen (G-I). Other labels:sm, stria medullaris; mol, molecularlayer; SuM, supramammillary nucleus;LM, lateral mammillary nucleus; ML,medial mammillary nucleus; Arc, arcuatehypothalamic nucleus; s, spine; den, den-drite. Arrows identify immunoreactivemedium-sized striatal neurons in A, aGolgi cell in the granule-cell layer of thecerebellum in E, reaction product at apostsynaptic site in a striatal dendrite inH, and reaction product localized to theneck of a spine arising from a dendrite inI. [Bars = 100 Am (A, C, and E), 1.0 mm(B, D, and F), or 2.0 ,Am (G-1).]

stria terminalis (Fig. 3C), the cingulum, and portions of thefornix, anterior commissure, and corpus callosum.

DISCUSSIONProtein Recognition by the Antiserum in Relation to Can-

didate Molecular Sizes of Di Receptors Found in Brain Mem-branes. The antiserum (i) immunoprecipitated a D1 receptorprotein fragment translated in vitro and (ii) detected twoproteins of 50 and 75 kDa on Western blots of striatal andhippocampal membrane extracts. The 50-kDa protein is in theapproximate range of the predicted molecular size encodedby the D1 receptor (13, 17, 18) and ofa protein found in striatal

I

membranes to have the expected pharmacological specifici-ties for the D1 receptor (27). The 75-kDa protein is in therange of molecular mass of the glycosylated membrane-bound receptor protein (27-29). [A 62-kDa size of the D1receptor has also been reported (27).] Thus, our antiserummost likely recognizes both the membrane-inserted glycosy-lated D1 receptor and a smaller form also present in solubi-lized membranes.Our findings provide direct evidence with immunohis-

tochemistry that D1 receptors are widely distributed through-out the brain. The variation seen in the subcellular distribu-tion of immunoreactivity in different neuronal groups sug-

FIG. 4. (A) An axon terminal withtyrosine hydroxylase immunoreactivity

is_v i forms symmetric synapses at arrows witha dendrite (den) in the reticularnucleus ofthe thalamus. (B) Immunoreactive D1re-

ceptor in the neostriatum is localized to asmall axon terminal which forms anasymmetric synapse with spine (s) atringed arrow; an unlabeled axon aboveforms an asymmetric synapse at arrowwith another spine (s). (C) D1 receptorimmunoreactivity is localized to myeli-nated (mye) and unmyelinated axons (at

arrows) in the substantia nigra pars re-

ticulata. (Bars = 2.0Anm.)

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gests that D1 receptor-expressing cells may vary greatly intheir production, transport, and localization of receptor pro-tein. Moreover, the prevalence of fiber bundle and axonterminal labeling in some regions suggests that the axonaltransport and presynaptic localization of these receptors maycontribute significantly to their functional role in dopamineregulation.

Correlation with Previous Studies ofD1 Receptor Lclzationand Functional Significance. Our immunohistochemical resultsare consistent with the known organization of dopamine-containing neurons and their projections (30) and provideevidence that D1 receptors mediate the action of dopamine inall dopamine projection pathways, including mesolimbic, ni-grostriatal, mesocortical, incertohypothalamic, tuberohy-pophysial, and tuberoinfundibular systems. The results alsoagree with previous studies where ligand binding (12, 31-33)and mRNA expression (13, 31, 34) have been used to map D1receptors and with the localization of DARPP-32 (35), aphosphoprotein enriched in neurons with Di receptors.The high concentration of immunoreactive D1 receptors in

the limbic system (cingulate, piriform, and entorhinal cortices;septal nucleus; hippocampus; amygdala; mammillary com-plex; medial dorsal nucleus) and in the fiber bundles connect-ing these areas (cingulum, fornix, stria terminalis) suggeststhat this receptor protein may be very important in mediatingfunctions associated with memory, learning, and cognitiveprocessing. Despite the low to moderate levels of D1 receptorbinding reported in many ofthese areas (12), a recent study hasshown that limbic forebrain neurons exhibit high levels of D1receptor mRNA (14). The functional significance of the highD1 receptor densities seen in the membranes of cortical andhippocampal pyramidal neurons is unclear.The reticular nucleus of the thalamus has not been previ-

ously reported as a site for dopamine innervation or dopaminereceptor localization. This nucleus receives ascending inputfrom the basal forebrain and collateral projections from mostthalamocortical and corticothalamic pathways which traverseits borders en route to target sites (see ref. 36). The reticularnucleus regulates the rhythmicity of thalamocortical neuronsin different states of arousal (37), thereby influencing higher-order cognitive processing. One study has shown abnormali-ties in the reticular nucleus in Alzheimer disease (36).As expected, D1 receptor immunoreactivity in the basal

ganglia was high, and the frequency of labeled striatal neu-rons (about half of striatal cells) is in accord with a previousreport ofmRNA localization (34). The postsynaptic localiza-tion ofD1 receptors in striatal dendrites and spines, especiallyspine necks, concurs with ultrastructural studies ofdopamineinnervation of striatal neuropil (38, 39). The presence of D1receptor-containing axons in the pars reticulata of the sub-stantia nigra provides direct evidence to support biochemicaldata that D1 receptors are localized presynaptically to stria-tonigral projection neurons (40). The immunoreactivity foundin axons within the striatum and in dendrites of the parsreticulata was unexpected and suggests that D1 receptorsengage in other neuronal circuits which mediate dopamineactivity within the basal ganglia.Our findings demonstrate the feasibility of obtaining signif-

icantly increased cellular resolution of D1 receptors. Double-labeling methods can now be applied to determine the neuro-transmitters and signal transduction pathways in D1 receptor-bearing neurons, the membrane localization ofthese receptorsin relation to dopaminergic inputs, and the colocalization ofD1receptors with other receptor proteins. It will also be possibleto define the specific circuits in which D1 receptors functionpresynaptically, postsynaptically, and nonsynaptically in dif-ferent dopaminergic projection pathways.

This research was supported in part by National Institutes ofHealth Grant NS16367 (M.D. and J.F.G.) and National Science

Foundation Grant BNS8819989 (N.A.). We thank Robert Woodland,Jack L. Leonard, Dan Kelleher, and Madelyn R. Schmidt (Univer-sity of Massachusetts Medical School) for their help.

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