DOPAMINE D5 RECEPTORS OF RAT AND HUMAN BRAIN

Z. U. KHAN,* A. GUTIERREZ, R. MARTıN, A. PENAFIEL, A. RIVERA and A. DE LA CALLEDepartment of Cell Biology, Faculty of Sciences, University of Ma´laga, Teatinos 29071, Ma´laga, Spain

Abstract—In contrast to dopamine D1 receptors, the anatomical distribution of D5 receptors in the CNS is poorly described.Therefore, we have studied the localization of dopamine D5 receptors in the brain of rat and human using our newly preparedsubtype-specific antibody. Western blot analysis of brain tissues and membranes of cDNA transfected cells, and immunoprecipita-tion of brain dopamine receptors suggest that this antibody is highly selective for native dopamine D5 receptors. The D5 antibodylabeled dopaminergic neurons of mesencephalon, and cortical and subcortical structures. In neostriatum, the D5 receptors werelocalized in the medium spiny neurons and large cholinergic interneurons. The D5 labeling in caudate nucleus was predominantlyin spines of the projection neurons that were frequently making asymmetric synapses. Occasionally, the D5 receptors were alsofound at the symmetric synapses. Within the cerebral cortex and hippocampus, D5 antibody labeling was prominent in thepyramidal cells and their dendrites. Dopamine D5 receptors were also prominent in the cerebellum, where dopamine innervationis known to be very modest. Differences in the localization of D5 receptors between both species were generally indistinguishableexcept in hippocampus. In rat, the hippocampal D5 receptor was concentrated in the cell body, whereas in human it was alsoassociated with dendrites.

These results show that D5 receptors are localized in the substantia nigra-pars compacta, hypothalamus, striatum, cerebral cortex,nucleus accumbens and olfactory tubercle. Furthermore, the presence of D5 receptors in the areas of dopamine pathways suggeststhat this receptor may participate actively in dopaminergic neurotransmission.q 2000 IBRO. Published by Elsevier Science Ltd.All rights reserved.

Key words:D1-like receptors, immunoblot, immunoprecipitation, immunocytochemistry, electron microscopy, immunofluorescense.

The dopamine D5 receptor is a member of the D1-like family,which consists of D1 and D5 subtypes.11 Because of theunavailability of specific agonists and antagonists, most ofthe pharmacological and physiological studies do not differ-entiate the individual role of D1 and D5 receptors. The onlymajor difference that has widely been recognized is that theD5 receptor displays higher affinity for the dopamine neuro-transmitter than the D1 subtype21,37,38which implies that theD5 subtype should participate more effectively in dopamin-ergic functions. However, it is the D1 subtype that has mostoften been described to be associated with D1-like functions.This understanding was derived from the finding that D1receptors are predominantly localized in the primary dopa-minergic projection areas, such as striatum and nucleusaccumbens. But growing evidence has begun to indicatethat both D1 and D5 receptors contribute individually indopaminergic neurotransmission. For example, Kimuraetal.29 have shown that D5 receptor coupling to the G-proteinis stronger than D1, which is consistent with the earlierobservation that D5 receptors have a higher affinity forthe dopamine neurotransmitter for D5 receptors. Further-more, blockade of D5, but not D1 dopamine receptor,suppresses reproductive behavior associated with D1agonist (non-selective for D1 and D5 receptor) stimulation.1

More recently, Dziewczapolskiet al.13 have shown thatcontralateral rotations induced by SKF 38393 (a D1-like

agonist) in 6-hydroxydopamine lesioned rats were completelyprevented by administration of an antisense oligodeoxy-nucleotide for the D1 receptor, whereas administration ofthe antisense oligodeoxynucleotide for D5 receptor poten-tiated the effect, indicating that D1 receptors play a facilita-tory role in locomotion and D5 exert an inhibitory effect.During development, both D1 and D5 receptors also playdifferent roles. Wanget al.39 have reported that Cajal-Retziusand migrating neurons were immunoreactive for D5 receptor,while radial glia cells were labeled with D1 receptor antibody.

The localization of the dopamine D5 receptor in rat2 andprimate4 brains has been reported. However, the antibodyused for the rat studies was not as rigorously tested for speci-ficity and selectivity as the one used for the primate brain. Inrat brain, the study also lacks thorough and complete informa-tion of D5 receptor distribution. Moreover, there are manydiscrepancies among the several studies reporting the distri-bution of D5 receptor mRNA in the CNS.3,9,21,25,32,33,37,38,40

Therefore, our aim here was to study the D5 receptor locali-zation sites and explore the differences between rat andhuman brain. To achieve this we have prepared subtype-specific and selective anti-peptide antibodies against D5receptor. An extensive characterization of the antibodieswas performed to demonstrate their specificity and selectivity.

EXPERIMENTAL PROCEDURES

Materials

SCH 23390, [N-methyl-3H] (81.4 Ci/mmol) was purchased fromDuPont-New England Nuclear (Wilmington, DE, USA). The (1)butaclamol-HCl was from Research Biochemicals Inc. (Natick, MA,USA). The crude membranes of recombinant Sf9 cells expressingdopaminergic human D1, D2L, D3rat, D4.2 and D5 receptors wereobtained from BioSignal, Inc. (Quebec, Canada). Peptides weresynthesized by the Core Facility at the School of Biological Sciences,University of Missouri-Kansas City (Kansas City, MO, USA).

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NeuroscienceVol. 100, No. 4, pp. 689–699, 2000q 2000 IBRO. Published by Elsevier Science Ltd

Printed in Great Britain. All rights reserved0306-4522/00 $20.00+0.00PII: S0306-4522(00)00274-8

Pergamon

www.elsevier.com/locate/neuroscience

*To whom correspondence should be addressed at: 333 Cedar Street,Section of Neurobiology, Yale School of Medicine, New Haven, CT06511, USA. Tel.:11-203-785-6493; fax:11-203-785-5263.

E-mail address:zkhan@kafka.med.yale.edu (Z. U. Khan).Abbreviations:KLH, keyhole limpet hemocyanin; LTP, long-term poten-

tiation; PAP, peroxidase anti-peroxidase; PBS, phosphate-bufferedsaline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis; TH, tyrosine hydroxylase.

All the studies on human subjects were conducted in accordancewith the Declaration of Helsinki. The protocols used with our studieswere approved by the appropriate animal care committee at Universityof Malaga. All efforts were made to minimize the number of animalused and their suffering.

Preparation of antibody to D5 receptor

A sequence common to both rat and human D5 receptor, peptideDEEEGPFDRMF (corresponding to residues 428–43821,37,38) wasobtained from the corresponding translated cDNA sequences andsynthesized. Computer assisted homology study showed that theselected amino acid sequence is exclusive for the dopamine D5 recep-tor subtype. An additional cysteine was added at the carboxy end of thepeptide for coupling to keyhole limpet hemocyanin (KLH) protein.Peptide-KLH conjugation and rabbit imunizations were done asdescribed earlier.27,28 A solid-phase enzyme-linked immunoassaywith immobilized synthetic peptides was used to monitor antibodyproduction. Affinity-purification of the antisera was done on the corre-sponding antigen peptide immobilized columns as described in detailelsewhere.27

Membrane preparation and solubilization

Cortices were collected from 10 adult Sprague–Dawley rat brainsafter decapitation and homogenized with 10% sucrose in 50 mM Tris-HCl, pH 7.4. The homogenate was centrifuged at 3000 rpm for 5 minand microsomal membranes were prepared as described earlier.27,28

The membranes were solubilized with 1% digitonin19 in 50 mMTris-HCl, pH 7.4 containing 1 mM EDTA, 5 mM KCl, 1.5 mMCaCl2, 4 mM MgCl2, 120 mM NaCl and 0.1% ascorbate for 30 minat 48C. After centrifugation at 29,000 rpm for 1 h, the supernatant wasused for immunoprecipitation and binding assays.

Immunoprecipitation of dopamine D5 receptors

The solubilized receptor membranes (800ml containing 150 fmol of[ 3H]SCH 23390 binding sites) were incubated overnight at 48C with 0–60ml of antisera to D5. The bound dopamine receptors, as receptor-antibody complexes, were separated after incubation with 80ml of40% (vol/vol) suspension of protein-A agarose followed by centrifu-gation. The non-immunoprecipitated receptors in supernatant wereassayed for [3H]SCH 23390 binding. For the binding, 500ml of super-natant was incubated with 2 nM of radioligand for 1 h at 228C in totalvolume of 1.0 ml of 50 mM Tris-HCl (pH 7.4) containing 1 mMEDTA, 5 mM KCl, 1.5 mM CaCl2, 4 mM MgCl2 and 120 mMNaCl. The reaction was stopped by rapid filtration through 0.3% poly-ethylenimine soaked Whatman GF/B filters7 and washed three timeswith 50 mM Tris-HCl, pH 7.4. Filters were dried and counted. Thenon-specific binding of the dopamine receptors was determined in thepresence of 2mM (1) butaclamol-HCl. The amount of immuno-precipitated receptors was calculated after subtracting the values ofsupernatant from total specific binding (100%) of [3H]SCH 23390.The 100% values are representative of 0ml of D5 antibody or 60mlpre-immune serum treated supernatant.

Immunoblots

Immunoblots were done according to Khanet al.26–28 Briefly, ratcortical and striatal membrane proteins (15–20mg/lane) were separ-ated by 10% sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS–PAGE) and transferred to nitrocellulose membranes.The transblotted nitrocellulose membranes were incubated with 5mg/mlof affinity-purified primary antibodies to D5 which were diluted inphosphate-buffered saline (PBS) containing 0.05% Tween-20 and1% bovine serum albumin, followed by anti-rabbit IgG (Sigma) diluted1:100 and peroxidase anti-peroxidase (PAP) complex (Sigma) diluted1:100. The polypeptide bands were visualized with peroxidase reaction.

For the immunoblots of membranes of the Sf9 cell lines expressingdopamine receptors, 4mg of total protein was electrophoresed on 10%SDS–PAGE and transblotted onto nitrocellulose membranes. Mem-branes were saturated by incubating with PBS containing 10% non-fat dry milk, 5% sheep serum, 5% donkey serum and 0.1% Tween-20for 2 h at room temperature, which was followed by incubation with5mg/ml antibodies to D5 for overnight at 48C. The secondary anti-body, anti-rabbit IgG-HRP (Amersham, Arlington Height, IL, USA)was diluted 1:2000 and incubated for 1 h. Immunoreactive bands were

developed by using ECL kit as instructed by manufacturer (Amersham,Arlington Height, IL, USA).

Immunocytochemistry

Twelve adult male Sprague–Dawley rats (Charles River, France) ofabout 250 g body weight were perfused transcardially with 400 ml of4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. Brains werethen dissected out and postfixed in the same fixative for 3 h and cryo-protected with 30% sucrose. Thepost mortem(7–15 h intervals)human brain tissues were obtained from clinical autopsy at CarlosHaya Hospital at Ma´laga (Malaga, Spain). These tissues were collectedfrom five males of 35–50-years-old without neurological or psychia-tric disorders. Human brain blocks of 5 mm2 from motor cortex (area4), visual cortex (area 17), prefrontal cortex (area 46), cerebellum,hippocampus and striatum were immersed in 4% paraformaldehydein phosphate buffer, pH 7.4 for 48 h at 48C. Blocks of human and ratbrains were cut with freezing microtome (30mm) or a Vibratome(50mm) and processed for light and electron microscopic levelstudies.

Free-floating sections were processed for light microscopic levelstudies as described earlier.28 All antibodies were diluted in 0.1 MPBS with 0.3% Triton X-100. Sections were incubated for two daysat 48C in primary affinity-purified antibody to D5 (15mg/ml) whichwas followed by incubation in the secondary antibody sheep anti-rabbitIgG (1:100; Sigma) or biotinylated goat anti-rabbit antibody (1:200;Jackson) and then in rabbit PAP complex (1:100, Sigma) or ABC Elitekit (1:100; Vector). The reaction was developed by using either 0.05%diaminobenzidine and 0.01% hydrogen peroxide or diaminobenzidine-glucose oxidase reaction.

For double-label immunofluorescense studies, sections were incu-bated with D5 antibody and mouse monoclonal tyrosine hydroxylase(TH) antibody (1:1000; Chemicon) followed by incubation with goatanti-rabbit IgG-Cy3 (1:400; Jackson) and goat anti-mouse IgG-FITC(1:100, Jackson).

For electron microscopic level analysis, Vibratome sections wereincubated with D5 antibody and visualized by the immunoperoxidasemethod using ABC Elite kit. Sections were osmicated, dehydrated, andflat embedded in Durcupan ACM (Fluka). The resin embedded sectionswere cut into ultrathin sections on an ultramicrotome (Reichert) andobserved in JEOL electron microscope.

RESULTS

Controls

In control experiments of western blots (Fig. 1B), immuno-precipitation and brain sections (Fig. 1C), we found that theD5 immunoreactivities were abolished by either preincuba-tion of antibody with cognate peptide or incubation with pre-immune sera. Furthermore, in immunoprecipitation experi-ments we observed no [3H]SCH 23390 binding activitywhen an unrelated antibody, anti-b of the GABAA receptor(bd 17 from Boehringer) was used, suggesting a specificimmunoreaction of D5 antibody.

Specificity and selectivity of the dopamine D5 receptor antibody

The specificity and selectivity of the antibody was tested byimmunoprecipitation of the solubilized native dopaminereceptors, and immunoblots of tissue membranes and Sf9cell membranes transfected with cDNA of dopamine recep-tors, which are shown in Fig. 1A and B. The antibodyshowed a concentration dependent immunoprecipitationof [3H]SCH 23390 binding sites from solubilized extractof rat cerebral cortex. The saturation point was observed at20ml and maintained until 60ml (Fig. 1A), indicating thesensitivity and specificity of the D5 antibody. The antibodywas able to immunoprecipitate 34.34.8% of the totalbinding sites.

In both cortical and striatal tissues of rat brain, antibody to

Z. U. Khanet al.690

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Fig. 1. Characterization of D5 antibody by immunoprecipitation (A), immunoblots (B) and immunocytochemistry (C) studies. (A) shows D5 antibodyconcentration-dependent precipitation of D5 receptors as [3H]SCH 23390 binding sites from rat cerebral cortex, which reached to saturation at 20ml. Theantibody was able to immunoprecipitate 34.3^4.8% (B) native D5 receptor. The remaining non-immunoprecipitated (X) binding sites may represent mostlyD1 receptor and some serotonin receptor. Data represented are mean^S.E.M. of three experiments, each done in triplicate. (B) shows the reactivity of singlepolypeptide band of 47,000 mol. wt in both rat cerebral cortex and striatum, which was abolished by pre-absorption of the D5 antibody with cognate peptide.However, the right panel shows the specific reactivity of antibody with D5 receptor containing recombinant cells and not with cell lines expressing severalother dopamine receptor subtypes. (C) shows control immunocytochemistry of sagittal brain sections from rat cerebellum (i) and hippocampus (ii), and humancortex area 17 (iii) with D5 antibody preabsorbed with cognate peptide. M, molecular layer; P, Purkinje cell layer; G, granule cell layer;I–V, indicate the

cortical layers. Scale bar�91mm.

the D5 receptors immunoreacted with a single protein band of47,000 mol. wt (Fig. 1B), the expected molecular size aspredicted from molecular cloning.21,37,38Furthermore, in celllines expressing dopamine receptors, the D5 antibody bindsselectively to the recombinant cells transfected with D5cDNA and showed no reactivity with cells expressing othermembers of the dopamine receptor family (Fig. 1B). Theseresults demonstrate that this antibody is selective for D5receptors.

Taken together these results suggest that this antibodyis subtype-specific and selective for native dopamine D5receptors.

Expression of the D5 receptor in midbrain

The D5 antibody showed immunoreactivity in the cellbodies of substantia nigra-pars compacta and also in parsreticulata (Fig. 2A, B). Double immunofluorescense studyof D5 antibody and TH, a marker for dopaminergic cells,showed most of the neurons expressing D5 receptors are

TH-positive (long tailed arrows in Fig. 2C and D). Indeed,we also observed several non-dopaminergic D5 positiveneurons, most probably GABAergic (short tailed arrows inFig. 2E and F). At electron microscopy level, the D5 immuno-reactivity was often observed in neuronal dendrites, whichwere making asymmetric synapses with axons (Fig. 3A andB). This reactivity was associated with the plasma membranesof the neurons. Occasional presynaptic D5 receptors were alsoseen (Fig. 3A).

Strong labeling of D5 antibody was found in the inferiorcolliculus (Fig. 4A). Numerous cell bodies and their initialprocesses could be seen in dorsal and external cortices andcentral nucleus. The cells of the oculomotor nucleus and deepgrey layers of superior colliculus were also immunostained(not shown).

Localization of the D5 receptor in hypothalamus andthalamus

Weak immunostaining of the D5 antibody was observed in

Z. U. Khanet al.692

Fig. 2. Immunostaining of dopamine D5 receptor in substantia nigra of rat brain. Parasagittal sections in (A) and (B) show that the D5 receptors are localized inboth the substantia nigra pars compacta (arrows in A) and pars reticulata (arrows in B). (C–F) show the double immunofluorescence labeling of D5 receptorsas Cy3 (red), and tyrosine hydroxylase (TH) (marker for dopaminergic cells) as FITC (green). Long tail arrows indicate the colocalization of D5 in TH-positive dopaminergic neurons, although, not all the TH-positive cells were labeled with D5 receptor (arrowhead). There were also D5 labeling in thenon-dopaminergic cells, which are most probably GABAergic as shown by small tail arrows. Arrowhead in (F) indicates the TH immunopositive cell, which is notlabeled with D5 antibody indicating the specificity of immunoreaction. PC, substantia nigra pars compacta; PR, substantia nigra pars reticulata. Scale

bars�400mm (A), 75mm (B), 33mm (C–F).

the neurons of hypothalamic arcuate (Fig. 4B), mammillary(Fig. 4C) and supraoptic nuclei. However, a strong labelingof D5 receptor was seen in the thalamus (Fig. 4D–F). Reticularnucleus can be observed with a moderate immunostaining

(Fig. 4D). Thalamic structures showed perikaryal labeling(Fig. 4E, F), which was dominantly present in lateral dorsal,anterior ventrolateral, anterior dorsomedial and lateral posteriorthalamic nuclei.

Antibodies to dopamine D5 receptors 693

Fig. 3. Electron micrograph of pars reticulata (A) and pars compacta (B) of substantia nigra from rat brain showing labeling of D5 receptors (arrowheads) indendrites (d), which are making asymmetric synapse (arrow) with D5 labeled (an) and unlabeled (a) axons. Scale bar�200 nm.

Fig. 4. Dopamine D5 receptor immunostaining in the sagittal sections of rat brain inferior colliculus (A), hypothalamic arcuate (B) and mammillary nuclei (C),thalamus (D–F), reticular nucleus (Rt; D) and globus pallidus (GP; D). Most of the labeling is associated with perikarya. LD, laterodorsal thalamic nucleus;LP, lateral posterior thalamic nucleus; VL, ventrolateral thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic

nucleus. Scale bars�83mm (A), 200mm (B, C), 400mm (D), 100mm (E, F).

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Fig. 5. Localization of D5 receptors in sagittal sections of subcortical forebrain areas of rat and human brains. Low magnification picture of rat striatumimmunostained for D5 receptors (A), which at higher magnification (B) shows that labeling is in small size neurons (arrowheads). Strongly labeled largeneurons (indicated with arrows), probably cholinergic, were also observed. However, in human striatum (C) in addition to labeling of cells, strong D5 stainingwas also found in dendritic fibres (arrows). (D) shows the neurofibre staining in human striatum. Neuropil immunostaining of D5 receptors was seen inaccumbens nucleus (NAc; E) and islands of calleja (ICj; E and F). Tu, olfactory tubercle; VP, ventral pallidum. Scale bars�333mm (A), 35mm (B), 62.5mm

(C), 20mm (D), 267mm (E, F).

D5 receptors in subcortical forebrain areas

In striatum, both rat and human brains were used for study.In rat, the D5 receptor was localized in the striatal medium-sized spiny neurons and large cholinergic neurons (Fig. 5Aand B). However, in human, the labeling was mostly asso-ciated with the extended long dendrites (Fig. 5C and D). Instriatum, at electron microscopic level, we found that D5receptors were localized in the dendrites and spines (Fig.6A). Often asymmetric synapses were formed on D5 con-taining spines. Occasional symmetric synapses were alsoobserved. In accumbens nucleus, immunolabeling wasfound mainly in the neuropil (Fig. 5E). D5 receptors werealso seen in the globus pallidus (Fig. 4D), islands of Calleja(Fig. 5F), olfactory tubercle and septal area.

D5 receptors in the hippocampus

In hippocampal formation, both rat and human brains wereused for immunostaining studies. In rat, strong labeling of theD5 receptor was found in the pyramidal cells of hippocampusproper (Fig. 7D and F). However, in human, pyramidal cellbodies showed low labeling. Instead, their apical dendriteswere more strongly stained (Fig. 7A). Human brains showedlow staining in the CA1 region. In dentate gyrus of rat, D5immunoreactivity was observed in the granule cell bodies(Fig. 7E), whereas in human, it was in the fibres of the mole-cular layer (Fig. 7C), presumably originating from the granulecells. The neuronal cell bodies of the dentate gyrus showedstrong labeling and some of these cells were located at theborder of granule cell layer and hilus (Fig. 7E). Subicularregion of both the species was immunolabeled but showedsimilar differences as in other parts of hippocampus. In rat, D5

receptor was concentrated in neuronal cell bodies but inhuman it was also in their dendrites (Fig. 7B).

Localization of D5 receptor in the cerebral cortex, cerebel-lum and olfactory bulb

The study in cerebral cortex includes rat and several areasof human brains. D5 receptor was highly expressed in thepyramidal neurons and their dendrites (Fig. 8A–D), althoughwe have also observed occasional non-pyramidal cells. In rat,a prominent immunoreactive neuronal band was located inlayers IV–VI of frontal and limbic cortical areas (Fig. 8Cand D). This band was less dense in occipital cortex. Thecell body and apical dendrite of numerous pyramidal neuronswere strongly labeled (Fig. 8D). In human, the labelingpattern was similar to the rat. Areas 17 and 4 showed thestrongest immunoreactivity, whereas it was moderate to lowin areas 1, 2 and 46. An immunoreactive cell band was alsoobserved in all of the above areas in layersIV–VI (Fig. 8A andB). The electron microscopic analysis of area 17 from humanbrain indicates that D5 receptors are present in the spines, asite where asymmetric synapses are formed with afferentaxons (Fig. 6B, C).

In human cerebellum, the dendritic tree of Purkinje cellsshowed a moderate immunoreactivity for the D5 receptor(Fig. 8E). However, occasional strongly labeled dendriteswere also observed (arrows in Fig. 8E). The granule celllayer was not labeled. However in rat, apart from the Purkinjecells and their dendrites, a low reactivity was also seen in thegranule cell layer (Fig. 8F).

The D5 immunoreactivity was mostly associated with theexternal plexiform and mitral cell layers of olfactory bulb

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Fig. 6. Electron micrograph of the rat striatum (A) and human cortical area 17 (B and C) showing that the D5 receptors are localized in the dendrite (d) andspines (s), which are receiving asymmetric (solid arrows) as well as symmetric (open arrows) synaptic inputs from axon (a) of different origins. sn is unlabeled

spine. Scale bar�200 nm.

(Fig. 8G) and granule cell layer of accessory olfactory nucleus(Fig. 8H).

DISCUSSION

The localization of D5 receptor in the dopaminergic projec-tion neurons of substantia nigra-pars compacta was a surpriseto us because earlier immunohistochemical studies in rat2 andmonkey4 were unable to detect this. However, the presence ofD5 mRNA transcript3,9 has been reported in different subareasof substantia nigra. The localization of D5 receptors in dopa-minergic neurons suggests that this receptor may participatein the events of the nigrostriatal pathway. In rat, behavioral

and neurochemical recovery from partial 6-hydroxydopaminelesions of substantia nigra was blocked by treatment withSCH 2339014 (an antagonist of both D1 and D5 receptors).This observation indicates a role for the D1/D5 dopaminereceptor in the development of compensatory changes in thedopaminergic neurons. D1 receptors are predominantlyexpressed in the neuropil of pars reticulata,4 whereas D5receptors are localized in dopaminergic neurons of parscompacta. Therefore, the blocking effect produced by treat-ment with D1/D5 antagonists may primarily be due to thecontribution of D5 receptor function. D5 receptors werealso found in the substantia nigra-pars reticulata, an areawhich receives major input from the striatum via direct and

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Fig. 7. Immunoreactivity to the dopamine D5 receptors in sagittal sections of hippocampus. The CA2 field exhibits higher expression of D5 receptors thanCA1 in human brains (A). In rat this protein was equally strong in both CA1and CA2 areas (D). In rat, the immunoreactivity was mostly associated with thepyramidal cells (F), not in their dendrites (indicated with arrow), whereas in human it was also in dendritic fibres. Human subicular neurons and theirdendritesshowed moderate reactivity (arrows in B). (C) shows neurofibre staining in the human dentate gyrus (C). In hilus (Hil) and granular layer (G) of dentategyrus(DG) of rat brain, D5 receptors were present in the numerous cells (E), where several cells were observed attached with the border of granular layer andhilus

(indicated with arrows). M, molecular layer; P, pyramidal cell layer. Scale bars�200mm (A–C), 133mm (D), 67mm (E, F).

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Fig. 8. Dopamine D5 receptors in the sagittal sections of cerebral cortex, cerebellum, olfactory bulb and accessory olfactory nucleus. Pyramidal neurons withextended dendrites were found in area 17 of human cerebral cortex (A and B) as well as in frontal cortex of rat brains (C and D). Large cells of layers IV–VIwere intensely labeled. In human, layers II to III staining was mostly seen in dendritic fibres as shown by arrows (A). However, in rat it was in both neurons andfibres (C). In human (E) as well as rat (F), the majority of cerebellar D5 receptors were associated with dendrites of Purkinje cells and to a lesser extent to thePurkinje cell bodies. Granular cell layer of the rat cerebellum was labeled, but in human it was not. The staining of D5 receptors was observed in the externalplexiform layer (EPL), mitral cell layer (Mi) of olfactory bulb (G) and granular layer (GrA) of accessory olfactory nucleus (H).I–V, indicate the corticallayers, M, molecular layer; P, Purkinje cell layer; G, granular layer; GL, glomerular layer, IPL, internal plexiform layer; IGr, internal granular layer. Scale

bars�200mm (A), 100mm (B), 50mm (C), 33mm (D), 154mm (E), 63mm (F), 182mm (G), 91mm (H).

indirect pathways.43 The electron microscopic studies of thesubstantia nigra-pars compacta showed that the D5 receptors,which are frequently associated with dendrites, make asym-metric synapses with axons. The presynaptic localization ofthe D5 receptor in pars reticulata indicates that D5-labeledaxons may be originating from striatum and coursing throughthe striatonigral system. Apart from pars compacta ofsubstantia nigra, the other major dopaminergic neurons areassociated with hypothalamus. The localization of D5 recep-tor in hypothalamic arcuate nucleus suggests its role in theregulation of pituitary functions.1

D5 receptor was found in both medium spiny GABAergicand large cholinergic interneurons in rat and human striatum,which is consistent with an earlier report4 in monkey brain.The stimulation of striatal GABAergic neurons appears toincrease the release of GABA neurotransmitter from theaxon terminals.30 However, the localization of the D5 recep-tor in cholinergic interneurons suggests a role of D5 receptorin modulation of axonal input as well as in release of acetyl-choline from these neurons.8 The activation of dopamine D5receptors in striatal cholinergic interneurons are shown toenhance the Zn211 sensitive GABAA currents,42 indicatingthe participation of the D5 receptor in modulating GABAneurotransmission. In electron microscopic studies, the obser-vation that D5 receptor immunoreactive dendritic spines werepredominantly making asymmetric synapses with axonsprovides further evidence of D5 receptor involvement in themodulation of incoming excitatory cortical information.23

Another prominent dopaminergic target area is the cerebralcortex, where dopamine regulates both pyramidal and localcircuit GABAergic neuronal functions. The mesocorticaldopamine innervations to the prefrontal, cingulate and ento-rhinal cortices are involved in emotional, motivational andcognitive functions.20 Immunolabeling for D5 receptors wasobserved predominantly in layersIV–VI of the cerebralcortex, which is in agreement with other reports.2,4 In ratand human, the D5 receptor was mainly associated with pyra-midal neurons and their dendrites. Dendritic spines of thecortical pyramidal cells are major postsynaptic targets ofglutamatergic inputs.12 Dopamine terminals in the cortexpredominantly make synapses on spines of pyramidal cells36

suggesting that these D5 receptor containing pyramidal

neurons may interact with dopamine directly and modulatethe excitability of neurons, similar to the D1 receptor.18

The prominent species difference in D5 receptor label-ing was observed in the hippocampal formation. In rat,receptors were concentrated in the neuronal cell body,whereas in human, reactivity was also associated with thedendritic fibres of neurons. Despite the discrepancies inearlier reports about the localization of D5 receptors inbrain areas,2–4,9,10,21,25,32,33,37,38,40all have reported that hippo-campus is one of the areas where D5 receptor is dominantlyexpressed. The crucial role of the hippocampal dopaminergicsystem has been shown in several types of learning includingpassive avoidance,6 visual discrimination22 and positive re-inforcement learning.35 Moreover, dopamine depletion in thehippocampus impairs spatial navigation.16 Several groupshave shown that long-term potentiation (LTP), one form ofthe synaptic plasticity, is facilitated by the D1/D5 dopaminereceptors.15,24,34 A recent demonstration of direct protein–protein interaction between dopamine D5 and GABAA recep-tors in hippocampus31 suggests a novel mechanism for a D5receptor role in the modulation of other receptor functions.

CONCLUSION

The localization of D5 receptors in the cortical, subcorticaland limbic areas, where major dopaminergic innervationoccurs, indicates that this receptor is probably involved inthe enforcement of several physiological functions governedby the dopamine system. The D1-like (cumulative D1 andD5) receptors have been shown to be involved in severalphysiological functions including working memory,17,18,41

neural plasticity,13 early LTP,34 protein synthesis-dependentcomponent of late LTP,24 and induction of gene expression.5

However, the individual contribution of D5 receptors in thesefunctions is yet to be explored.

Acknowledgements—This study was supported by Spanish grantsDGICYT (PB94-0219-C02), DGESIC (PM98-0223) and Junta deAndalucia (CTS-0161). We would like to extend our thanks to MariaJoseLopez-Alvarez for her assistance with the immunocytochemicalexperiments. During this research, Z. U. Khan has received fellowshipsfrom Junta de Andalucia, Universidad de Ma´laga and European UnionBIOMED I.

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(Accepted6 June2000)

Antibodies to dopamine D5 receptors 699