microanatomical localization of dopamine receptor protein immunoreactivity in the rat cerebellar...

Ž .Brain Research 854 2000 130–138www.elsevier.comrlocaterbres

Research report

Microanatomical localization of dopamine receptor protein immunoreactivityin the rat cerebellar cortex

Paolo Barili a, Elena Bronzetti b, Alberto Ricci b, Damiano Zaccheo c, Francesco Amenta a,)

a Sezione di Anatomia Umana, Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, UniÕersita di Camerino, Via Scalzino, 3, I-62032`Camerino, Italy

b Dipartimento di Scienze CardioÕascolari e Respiratorie, UniÕersita ‘‘La Sapienza’’, 00161 Rome, Italy`c Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale, UniÕersita di GenoÕa, Genoa, Italy`

Accepted 2 November 1999

Abstract

Ž .Dopamine DA receptor subtype localization was investigated in rat cerebellar cortex using immunohistochemical techniques withantibodies raised against D1–D5 receptor protein. A faint D1 receptor protein immunoreactivity was developed in molecular and Purkinjeneurons layers. D2 receptor protein immunoreactivity was found primarily in cerebellar white matter followed by molecular and granularlayers and Purkinje neurons. Antibodies against D2S receptor protein were localized in molecular layer and to a lesser extent, in granularlayer. A few Purkinje neurons displayed a faint D2S receptor protein immunoreactivity. D3 receptor protein immunoreactivity wasobserved primarily in molecular and in Purkinje neurons layers of lobules 9 and 10. A faint D3 receptor protein immunoreactivity wasalso localized in Purkinje neurons and to a lesser extent, in molecular and granular layers of cerebellar lobules 1–8. D4 receptor proteinimmunoreactivity was found in cerebellar white matter. A pale immunostaining was also visualized in molecular layer. D5 receptorprotein immunoreactivity was localized primarily in molecular and Purkinje neurons layers and to a lesser extent, in granular layer and inwhite matter. The above results indicate that rat cerebellar cortex expresses the DA receptor subtypes so far identified. Purkinje neurons,which are the only efferent neurons of cerebellum, are richest in DA receptor protein immunoreactivity. This suggests that dopaminergicneurotransmission may modulate efferent inputs from cerebellum. The localization of the majority of D2 and D4 and of a faint D5 proteinreceptor immunoreactivity in cerebellar white matter suggests that these receptors may be presynaptic and transported axonally. q 2000Elsevier Science B.V. All rights reserved.

Keywords: Cerebellar cortex; Dopamine receptor; Receptor subtype; Immunohistochemistry; Rat

1. Introduction

The mammalian cerebellar cortex is supplied with acatecholaminergic innervation demonstrated since severalyears. It includes a well-developed noradrenergic and a

w xlimited dopaminergic supply 30,34 . Until recently, theŽ .small amounts of cerebellar dopamine DA were consid-

w xered to represent a precursor of noradrenaline stores 30,34 .The first suggestions of the occurrence of an independentdopaminergic innervation of cerebellum derived from evi-dence that electrolytic lesions of dopaminergic cell groups

Žof the substantia nigra and ventral tegmental area A8, A9,.A10 caused a marked reduction of DA levels in the

w xcerebellum 20 . More recent immunohistochemical inves-

) Corresponding author. Fax: q0039-0737-630-618; e-mail:[email protected]

tigations have shown that cerebellar dopaminergic innerva-tion consists of thin and varicose DA-immunoreactive

w xfibers ending primarily in the molecular layer 30 .The cerebellar cortex of various mammals including

humans expresses DA receptors belonging to the D1-likeand D2-like receptor superfamilies. These receptors weredemonstrated using biochemical, pharmacological and au-

w xtoradiographic techniques 5,8,10,13,15,16,26,29,30 . De-tailed autoradiographic studies have shown that D1-likereceptors were located in Purkinje neurons and in themolecular layer of mouse, rat, guinea pig, cat, monkey and

w xhuman cerebellar cortex 10 . D2-like receptors were foundin Purkinje neurons and in the molecular layer of lobules 9

w xand 10 of rat cerebellar cortex 10 .Until a few years ago, DA receptors were considered to

consist of two receptors subtypes, namely D1 and D2receptors. The application of molecular biology to DA

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 02306-9

( )P. Barili et al.rBrain Research 854 2000 130–138 131

receptor research has shown the existence of at least fiveŽ .receptor subtypes named DA D1 or D1A in the rat , D2

Ž . Ž .D2L and D2S , D3, D4 and D5 or D1B in the ratw xreceptors 14,33 . D1 and D5 receptors belong to the

D1-like receptor superfamily, whereas D2, D3 and D4receptors belong to the D2-like receptor superfamilyw x14,18,33 . Analysis of DA D1-like receptor mRNA hasshown low to moderate levels of D1 receptor mRNAprimarily in the granular layer and in the anterior lobules

w xof cerebellum 21,25,27 , and very low levels of D5 recep-w xtor mRNA 3,21 . Analysis of DA D2-like receptors re-

vealed little or no signal D2 receptor mRNA primarily inw xthe granular layer 7,21 , low levels of D3 receptor mRNA

throughout cerebellar cortex and very high levels in Purk-w xinje neurons of lobule 10 7 . DA D4 receptor mRNA was

w xdetected in high concentrations in mouse 37 and in loww xlevels in rat cerebellum 4,28 .

Autoradiographic studies were unable to identify DAreceptor subtypes expressed by cerebellar cortex. This isdue to the lack of truly selective radioligands, although thepresence of DA D1B and D3 receptors was suggested by

w xrecent investigations 23,31,32 . The development of anti-DA receptor protein antibodies has enlarged our knowl-edge on the cellular distribution of DA receptor subtypesin different brain areas including cerebellar cortexw x2,3,12,16 . However, so far, no complete picture of theexpression of DA receptor subtypes in cerebellar cortex isavailable.

The present study was designed to assess the mi-croanatomical localization of DA receptor subtypes in

sections of rat cerebellar cortex using immunohistochemi-cal techniques with anti-DA receptor protein antibodies.DA receptors are defined according to the nomenclaturesuggested by Subcommittees of the International Union ofPharmacology Committee on Drug Classification and Re-

Ž . w xceptor Nomenclature NC-IUPHAR 1 .

2. Materials and methods

2.1. Anti-DA receptor protein antibodies

Anti-DA D1–D5 receptor antibodies were obtained fromcommercial sources. Producers and characteristics of theseantibodies are summarized in Table 1. Their specificity forcorresponding receptors was assessed by Western blottingŽ .see Section 3 using as receptor source membranes ob-tained from whole rat brain homogenates for DA D1, D2,D2S, D3 and D5 and membranes obtained from frontalcortex for D4 receptor.

2.2. Animals and tissue treatment

Ž .Male Wistar rats of 250–300 g of body weight ns8were anaesthetized with an i.p. injection of pentobarbital

Ž .sodium 50 mgrkg and perfused through the ascendingŽaorta with a 0.9% NaCl solution containing heparin 20

.international unitsr100 ml . This solution was replaced bya fixative solution of a 4% freshly prepared paraformal-

Ž .dehyde and 0.4% picric acid in phosphate buffer pH 6.9 .

Table 1Summary of Western blot analysis of anti-DA D1–D5 receptor protein antibodies using membranes of rat brainAA: amino acids; a.p.: antigen peptide; Cys: cisteine; i.h.: immuno-histochemistry.

Antibody, species and producer Catalogue No. and working dilution Peptide band Effect of pre-adsorption with antigen peptides

D1 Cat. No. 324390 50 kDa Abolished reactivity by D1 a.p.22AA 9–21 Cys Lot No. B20607

Ž .Rabbit 1:1000 WesternŽ .Calbiochem 1:5000 i.h.

D2 Cat. No. 324393 48 and 51 kDa Abolished reactivity by D2 a.p. and partly by D2S a.p.271AA Cys -272–282 Lot No. B20061


D2S Cat. No. 324396 48 kDa Abolished reactivity by D2s a.p.247AA 239–246-Cys Lot No. B17549


D3 Cat. No. 324402 49 kDa Abolished reactivity by D3 a.p.11AA 2–10 Cys Lot No. B23335


D4 Cat. No. 324405 40–42 kDa Abolished reactivity by D4 a.p.187AA175–186-Cys Lot No. B17850

Ž .Rabbit 1:500 WesternŽ .Calbiochem 1:10,000 i.h.

D5 Cat. No. Sc. 1441 51 kDa Abolished reactivity by D5 a.p.Ž .AA 455–472 Goat 1:200 WesternŽ .Santa-Cruz 1:2500 i.h.

( )P. Barili et al.rBrain Research 854 2000 130–138132

At the end of perfusion, the skull was opened and the braintogether with the brainstem and the cerebellum were re-

moved. The brainstem and overlying cerebellum was de-tached from the rest of the brain.

Ž .Fig. 1. A Section of rat cerebellar cortex stained with Cresyl violet to verify microanatomical details. The arrow indicates Purkinje neurons layer; m:Ž .molecular layer; g: granular layer; w: white matter. B Section of rat cerebellar cortex exposed to anti-DA D1 receptor protein antibody. A faint

immunoreactivity was developed in the neuropil of molecular layer and within Purkinje neurons cell body. No reactivity was observed in the granular layerŽ .or in cerebellar white matter. C Section of rat cerebellar cortex exposed to anti-D1 receptor protein antibody pre-adsorbed with the corresponding

Ž .receptor blocking peptide. Pre-adsorption of antibody resulted in the disappearance of immune reaction and in the development of background. D Sectionof rat cerebellar cortex exposed to anti-D2 receptor protein antibody. Immunoreactivity was developed primarily in the white matter and to a lesser extent,

Ž .in molecular and granular layers. Only a few Purkinje neurons displayed D2 receptor protein immunoreactivity. E Section of rat cerebellar cortex exposedŽ .to anti-D2 receptor protein antibody pre-adsorbed with the corresponding receptor blocking peptide. F Section of rat cerebellar cortex exposed to anti-D2

receptor protein antibody pre-adsorbed with DA D2S receptor blocking peptide. Pre-adsorption with D2S receptor blocking peptide decreased to a someŽ .extent but did not abolish D2 receptor protein immunoreactivity indicating a cross-reactivity between the two antisera. G Section of rat cerebellar cortex

Ž .exposed to anti-D2S receptor protein antibody. Immunoreactivity was developed within granular layer and basket and stellate neurons small arrows ofŽ .molecular layer. Purkinje neurons displayed a faint D2S receptor immunoreactivity. H Section of rat cerebellar cortex exposed to anti-D2S receptor

protein antibody pre-adsorbed with the corresponding receptor blocking peptide. The significance of abbreviations in pictures B–H is the same as in pictureA. Calibration bars: picture A: 120 mm; pictures B–H: 50 mm.


Cerebellar blocks comprising all lobules were dissectedout, further fixed in the above solution for 4–6 h, putovernight in 15% sucrose at 48C and placed in a cryopro-

Ž .tectant medium OCT, Bio-Optica, Milan, Italy . OCTblocks were frozen in a dry ice–acetone mixture. Blockswere then cut serially using a y208C microtome cryostatand mounted on gelatin-coated microtome slides. Eachslide contained three 15 mm thick sections of cerebellum.

2.3. DA receptor protein immunohistochemistry

Groups of 10 slides per receptor investigated were used.The 1st section was stained with 0.5% cresyl violet to

Ž .verify microanatomical details Fig. 1A . The 2nd and 9thsections were exposed to the primary anti-DA receptor

Ž .antibody in phosphate buffer containing 0.2% wrv bovineŽ .serum albumin, 0.03% Triton X-100 and 0.1% wrv

sodium azide. Working dilutions of different antibodieswhich were assessed in a series of preliminary experimentsare detailed in Table 1. The 3rd section was incubated asabove with the primary antibody pre-adsorbed with itspeptide antigen at a dilution of 10 mgrml. Sections fromthe 4th to the 8th were exposed to primary antibodypre-adsorbed with peptide antigens used for obtaining otherDA receptor antibodies tested. The 10th section was ex-posed to a non-immune serum instead of the primaryantiserum. Incubation with antibody was accomplished in ahumid chamber at 48C for 12–18 h. Optimal antiseradilutions and incubation times were assessed in a series ofpreliminary experiments.

After incubation, slides were rinsed twice in phosphatebuffer and exposed for 30 min at 258C to biotinylatedanti-rabbit or anti-goat secondary antibodies at a dilutionof 1:200. The product of immune reaction was then re-vealed using the biotin–streptavidin immunostaining kitwith 0.05% 3,3-diaminobenzidine in 0.1% H O as a2 2

chromogen. Sections were then washed, dehydrated inethanol, mounted in a synthetic mounting medium andviewed at a light microscope equipped with both brightand dark-field condensers.

2.4. QuantitatiÕe analysis

The intensity of immunoreactivity developed in thedifferent layers of rat cerebellar cortex was assessed micro-densitometrically. Six sections of cerebellum per animalŽ .ns8 were examined at a final =200 magnification. Thesystem was calibrated taking as zero the background ob-tained in sections exposed to non-immune serum. Four 100mm2 areas of the molecular layer, Purkinje neurons layer,granular layer and white matter were then delineated ineach section by a measuring diaphragm. The intensity ofimmune staining was measured using a microdensitometry

Žprogram of an IAS 2000 image analyzer Delta Sistemi,.Rome, Italy connected via a TV camera to the micro-

scope.

2.5. Chemicals

Anti-DA D1–D5 receptor protein antibodies were ob-tained from the sources indicated in Table 1. D5 receptor

Ž .protein blocking peptide cat. No. 1441P was obtainedŽ .from Santa Cruz Biotechnology Santa Cruz, CA, USA .

The same peptide immunogens corresponding to thoseused for generating antibodies with the exception of theanti-D5 receptor antiserum were synthesized by the De-partment of Medicinal Chemistry of Camerino University.They corresponded to amino acids 9–21-Cys22 for the D1receptor of human origin, to amino acids Cys271-272–282for the D2 receptor of human origin, to amino acids2–10-Cys11 for the D3 receptor of human origin and toamino acids 176–185 for the D4 receptor of rat origin.Biotin–streptavidin immunostaining kit was a product of

Ž .Calbiochem-Novabiochem San Diego, CA, USA . OtherŽchemicals were purchased from Sigma St. Louis, MO,

.USA .

2.6. Statistics

Data of microdensitometric analysis of DA receptorprotein immunostaining in the different layers of rat cere-bellar cortex were analyzed statistically by analysis of

Ž .variance ANOVA followed by Duncan’s multiple rangetest as a post hoc test.

3. Results

The results of immunoblot analysis of anti-DA D1–D5receptor protein on membranes of rat brain are summa-rized in Table 1. As shown, Western blots revealed asingle band at approximately 50 kDa for anti-D1 receptorantibody. For anti-D2 receptor antibody two bands atapproximately 48 and 51 kDa were observed. A singleband at approximately 48 kDa was detected by analyzinganti-D2s receptor antibodies. Analysis of anti-D3 and -D4receptor antibodies revealed a single band at approxi-mately 49 kDa and 40–42 kDa, respectively. A singleband at approximately 51 kDa was found by analysis of

Ž .anti-D5 receptor antibody Table 1 . Pre-incubation ofantibodies with their respective peptides caused the com-plete disappearance of immune bands with the exceptionof the anti-D2 receptor antibody which reacted slightly

Žwith peptide used for generating the D2S receptor data.not shown .

Sections of rat cerebellar cortex exposed to antibodiesagainst DA receptor proteins developed a yellow-brown ordark-brown staining in the different layers of cerebellarcortex. Sections exposed to pre-immune serum or to anti-bodies pre-adsorbed with the corresponding receptor pro-tein peptide developed a yellow background only. The


pattern of distribution of immunoreactivity for each recep-tor protein is detailed below.

3.1. DA D1 receptor protein

A faint D1 receptor protein immunoreactivity was de-veloped in the neuropil of the molecular layer and in the

Ž .cell body of Purkinje neurons Fig. 1B . No reactivity wasobserved in the granular layer or in the cerebellar white

Ž .matter Fig. 1B . Exposure of sections to anti-D1 receptorprotein antibody pre-adsorbed with the corresponding re-ceptor blocking peptide resulted in the disappearance ofimmune reaction and in the development of background

Ž .only Fig. 1C . The use of anti-D1 receptor antibodypre-adsorbed with other receptor blocking peptides did notaffect the density and pattern of the above immunoreactiv-

Ž .ity data not shown . Data on the density of DA D1receptor immune reaction compared to other receptorsinvestigated are summarized in Table 1.


D2 receptor protein immunoreactivity was developedprimarily in cerebellar white matter and to a lesser extent,

Ž .in the molecular and granular layers Fig. 1D . In themolecular layer, immunoreactivity was localized both inthe neuropil and within basket and stellate neurons. Recep-tor protein immunoreactivity was also found in the cell

Ž .body of a small number of Purkinje neurons Fig. 1D .D2S receptor protein immunoreactivity was localized ingranular layer and neuropil of the molecular layer within

Ž .stellate neurons Fig. 1G . A few Purkinje neurons dis-played a faint D2S receptor protein immunoreactivity,whereas no immune staining was observed in white matterŽ .Fig. 1G . Exposure of sections to anti-DA D2 or D2Sreceptor protein antibody pre-adsorbed with the corre-sponding receptor blocking peptides caused the disappear-

Ž .ance of immune reaction Fig. 1E and H . The use ofanti-D2 or -D2S antibodies pre-adsorbed with other recep-

tor blocking peptides did not affect the density and patternof immune staining with the exception of a slight cross-re-activity of D2 antibody with D2S receptor blocking pep-

Ž .tide Fig. 1F . Data on the density of DA D2 and D2sreceptor immune reaction compared to other receptor sub-types investigated are summarized in Table 2.


D3 receptor protein immunoreactivity was observedprimarily in cerebellar lobules 9 and 10. In these structuresthe heaviest immunoreactivity was localized in the molecu-lar layer primarily within basket and stellate neurons and

Ž .to a lesser extent, in neuropil Fig. 2A . D3 receptorprotein immunoreactivity was also localized in the cell

Žbody of Purkinje neurons and in granular layer Fig. 2A.and Table 2 . In cerebellar lobules 1–8 a faint D3 receptor

protein immunoreactivity was observed in the cell body ofPurkinje neurons followed by granular layer and neuropil

Ž .of molecular layer Fig. 2C and Table 1 . Exposure ofsections to anti D3 receptor protein antibody pre-adsorbedwith the corresponding receptor blocking peptide resulted

Ž .in the disappearance of immune reaction Fig. 2B and D .Pre-absorption of DA D3 receptor protein with blockingpeptides corresponding to other receptor subtypes investi-

Ž .gated did not affect immunostaining data not shown .


Sections exposed to anti-D4 receptor protein antibodydeveloped an immunostaining primarily in cerebellar white

Ž .matter Fig. 2E and F . A faint immunoreactivity wasfound in molecular layer. Reaction was specific for D4receptor protein since immune reaction did not occur withantibody pre-adsorbed with D4 receptor blocking peptideŽ .Fig. 2G , but was unaffected by pre-adsorption of anti-body with blocking peptides corresponding to other DA

Ž .receptor subtypes data not shown . The relative distribu-tion of D4 receptor protein immunoreactivity in the differ-

Table 2Density of DA receptor protein immunoreactivity in the different layers of rat cerebellar cortexData are the mean"S.E. and represent the intensity of receptor protein immunostaining expressed in arbitrary units. For further details, see Section 2Ž .quantitative evaluation . Data of molecular layer indicate the intensity of staining in the neuropil of the molecular layer. Figures of Purkinje neuronsindicate the intensity of immune reaction in the cell body of Purkinje neurons. lob.: lobules; n.d.: not detectable.

Receptor Molecular layer Purkinje neurons Granule neurons layer White matter

D1 1.20"0.08 0.90"0.05 n.d. n.d.U Uaa aD2 3.95"0.09 2.04"0.19 3.98"0.18 4.95"0.48

Ua aD2S 3.25"0.18 0.95"0.06 2.21"0.20 n.d.UaŽ .D3 lob. 1–8 0.17"0.01 1.61"0.09 0.37"0.02 n.d.

UaŽ .D3 lob. 9–10 3.39"0.26 3.08"0.21 0.44" 0.03 n.d.aD4 0.23"0.02 n.d. n.d. 3.83"0.36

U Uaa a aD5 2.58"0.19 2.15"0.21 0.17"0.01 0.94"0.07

a p-0.01 vs. molecular layer.U

p-0.01 vs. Purkinje neurons layer.ap-0.01 vs. granule neurons layer.


Ž .Fig. 2. A Section of rat cerebellar cortex lobule 10 exposed to anti-DA D3 receptor protein antibody. Immunoreactivity was developed in the molecularŽ .layer primarily within basket and stellate neurons small arrows and to a lesser extent, in neuropil and within Purkinje neurons cell body. A less intense

Ž .immune reaction was found in granule neurons. B Section of rat cerebellar cortex lobule 10 exposed to anti-D3 receptor protein antibody pre-adsorbedwith the corresponding receptor blocking peptide. Pre-adsorption resulted in the disappearance of immune reaction and in the development of background.Ž .C Section of rat cerebellar cortex lobule 5 exposed to anti-D3 receptor protein antibody. A faint immunoreactivity was developed within Purkinje and to a

Ž .lesser extent, within granular neurons. D Section of rat cerebellar cortex lobule 5 exposed to anti-D3 receptor protein antibody pre-adsorbed with theŽ .corresponding receptor blocking peptide. E Section of rat cerebellar cortex exposed to D4 receptor protein antibody. A positive immunostaining was

Ž .observed in cerebellar white matter. A faint immunoreactivity was developed in the molecular layer. F Section of rat cerebellar cortex exposed to anti-D4receptor protein antibody pre-adsorbed with the corresponding receptor blocking peptide. For the significance of abbreviations, see legend to Fig. 1A.Calibration bars: 50 mm.

ent layers of cerebellar cortex compared to other receptorsis summarized in Table 2.


A moderate D5 receptor protein immunoreactivity wasobserved in the molecular layer both in stellate neurons

and in neuropil and within the cell body of PurkinjeŽ .neurons Fig. 3A . A faint immunoreactivity was also

Žobserved in white matter followed by granular layer Fig..3A and Table 2 . Exposure of sections to anti-D5 receptor

protein antibody pre-adsorbed with the corresponding re-ceptor blocking peptide caused the loss of immune staining


Ž .Fig. 3. A Section of rat cerebellar cortex exposed to anti-DA D5receptor protein antibody. Immunoreactivity was developed in stellateneurons and neuropil of molecular layer, in Purkinje neurons cell body

Ž .and to a lesser extent, in granule neurons and in white matter. B Sectionof rat cerebellar cortex exposed to anti-D5 receptor protein antibodypre-adsorbed with the corresponding receptor blocking peptide. Pre-ad-

Ž .sorption resulted in the disappearance of immune reaction. C Section ofrat cerebellar cortex exposed to anti-D5 receptor protein antibody pre-ad-sorbed with the D1 receptor blocking peptide. Pre-adsorption of antibodywith the D1 receptor blocking peptide did not affect immune reaction. Forthe significance of abbreviations, see legend to Fig. 1A. Calibration bar:50 mm.

Ž .Fig. 3B . The use of antibody pre-adsorbed with blockingŽ .peptides corresponding to D1 receptor protein Fig. 3C or

to other DA receptors investigated did not affect thedensity and distribution of DA D5 receptor immuno-

Ž .reactivity in the rat cerebellar cortex data not shown .

4. Discussion

The above results provide direct evidence that rat cere-bellar cortex expresses the five different subtypes of DAreceptors so far identified. As mentioned in Section 1,cerebellum is supplied with a dopaminergic innervationarising from dopaminergic cell groups of substantia nigra

w xand ventral tegmental area 17,30 . DA-containing im-munoreactive fibers were observed in the different layersof cerebellar cortex, with a greater accumulation in the

w x w xmolecular layer 30 . DA released in the cerebellum 11interacts with DA receptors which were demonstrated us-ing biochemical, pharmacological and autoradiographic

w xtechniques 5,8,10,13,19,22,26,29–32,39 . The main limitof these studies was in the inability to discriminate the DAreceptor subtype labeled due to lack of selective radioli-gands for the five DA receptor subtypes identified by

w xmolecular biology 14,33 . In this respect, the developmentŽ .of antibodies against D1-like D1 and D5 and D2-like

Ž .D2, D3 and D4 receptor proteins has contributed toincrease our knowledge on the dopaminergic system andhas enlarged our research capabilities on characterizationand localization of different DA receptor subtypesw x2,12,24 .

Anti-DA receptor protein antibodies were used for in-Ž . w xvestigating in the rat cerebellum D1 named D1A 2,16 ,

w x w x Ž . w xD3 2 , D4 4,12 and D5 named D1B 3 receptors.These studies have reported D1 receptor immunoreactivityin Golgi and in some Purkinje neurons, in the granular

w xlayer and in the neuropil of molecular layer 16 . Theoccurrence of D1 and D3 receptor protein immunoreactiv-ity was not confirmed in the cerebellum using the sameantibody dilution demonstrating immune reaction in other

w xbrain areas 2 . Studies on D4 receptor reported receptorprotein immunoreactivity in the molecular layer and in

w xstructures resembling climbing fibers 4 or in Purkinjeneurons, in stellate neurons of molecular layer as well as in

w xa few Golgi neurons of granular layer 12 . D5 receptorprotein immunoreactivity was found primarily within Purk-inje neurons and their tree in cerebellar vermis as well as

w xin granular layer 3 . As to date, no studies have comparedthe density and distribution of cerebellar DA receptorsubtypes, in the present work we have performed anextensive analysis of the expression of D1-D5 receptorsubtypes throughout rat cerebellar cortex. Anti-receptorprotein antibodies used were obtained from commercialsources. Data of immunoblotting analysis strongly suggestthat these antibodies were selective for the different recep-


tor subtypes investigated. The molecular size of proteinbands revealed by Western blot analysis is in agreement

w xwith the predicted size of 49 kDa for D1 receptor 36 , 47w x w xkDa for D2 receptor 9 ,49 kDa for D3 receptor 35 , 40w x w xkDa for D4 receptor 38 , 50–52 kDa 6 for D5 receptor.

The density of these sites was also compared in variouslayers of cerebellar cortex and in different portions ofcerebellum. Moreover, we have detailed for the first timethe distribution of DA D2 receptor in rat cerebellar cortex.

Comparatively, among D1-like receptors, the D5 sitewas the most expressed. The heaviest labeling occurred inthe cell body of Purkinje neurons and probably in theirdendrites, as inner portions of the molecular layer dis-played a moderate receptor protein immunoreactivity. Theobservation of D5 receptor protein immunoreactivity innerve fibers of the white matter of cerebellum confirms the

w xsuggestion of an axonal transport of D1-like receptors 16and indicates that these sites may have a presynapticlocalization. D2 receptor represents the D2-like receptorsubtype most largely represented in the cerebellar cortex,with a widespread distribution, including white matter. TheD2S isoform is located almost exclusively in cerebellargray matter. Further work is necessary to define the local-ization of the D2L receptor using antibodies raised againstthis receptor isoform.

D3 receptor is the receptor subtype most largely investi-w xgated in the cerebellum 2,23,31,32,39 , where it has prob-

w xably functional relevance 5 . Similarly as reported inw xliterature 2,23,31,32,39 , we have observed the greatest

density of D3 receptor protein immunoreactivity in lobules9 and 10. In these lobules, immunoreactivity was accumu-lated primarily within Purkinje neurons and their dendriteswhich are located in the molecular layer. A faint DA D3receptor protein immunoreactivity was also observed inother cerebellar lobules, where no presence of D3 receptorwas reported by previous autoradiography studiesw x23,31,32,39 . This apparent discrepancy is probably due tothe higher sensitivity of immunohistochemical detection ofDA receptor protein immunoreactivity in comparison withlight microscope autoradiography.

Data on D4 receptor protein localization are similarfrom those reporting a sparse immunoreactivity primarilyin the molecular layer associated with structures resem-

w xbling climbing fibers 4 , but are different from the find-ings of an intense labeling of Purkinje neurons, stellateneurons of molecular layer and Golgi neurons of granular

w xlayer reported by another group 12 . In our study, theabove structures displayed only a faint immunoreactivity,whereas we have observed immune staining of whitematter, which was apparently not analyzed by formerinvestigations. The sequence of receptor peptide used forgenerating anti-D4 receptor protein antibody was the same

w xin the present study and in the work of Defagot et al. 12 .Hence, we have no explanation for this apparent discrep-ancy. The observation of DA D4 receptor protein stainingin cerebellar white matter suggests that D4 receptor may

have a presynaptic localization and is transported alongnerve fibers. In line with this hypothesis are some prelimi-nary observations showing the loss of this immunoreactiv-ity after lesions of mesencephalic dopaminergic nucleiprojecting to the cerebellum. Further experiments are inprogress for detailing the origin and course of these projec-tions.

In conclusion, this study has shown that DA receptorsubtypes so-far characterized are located in the rat cerebel-lar cortex. The uneven distribution of immunoreactivity invarious layers of cerebellar cortex is probably related to adifferent meaning of receptor populations in cerebellarphysiology. A better understanding of the distribution ofthese sites may contribute to identify their role in normaland pathological conditions.

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