周里钢2002

PREPRODYNORPHIN-,PREPROENKEPHALIN-,PREPROTACHYKININ A-AND PREPROTACHYKININ B-IMMUNOREACTIVE NEURONS INTHE ACCUMBENS NUCLEUS AND OLFACTORY TUBERCLE:DOUBLE-IMMUNOFLUORESCENCE ANALYSIST.FURUTA,a L.ZHOU a and T.KANEKO a ;b ÃaDepartment of Morphological Brain Science,Graduate School of Medicine,Kyoto University,Kyoto 606-8501,JapanbCREST,JST (Japan Science and Technology),Kyoto 606-8501,JapanAbstract öPreprodynorphin (PPD),preproenkephalin (PPE)and preprotachykinins A (PPTA)and B (PPTB)are known to be expressed by neostriatal projection neurons.In the present study,we investigated the distributions and colocaliza-tions of immunoreactivities for those prepropeptides in the ventral striatum,such as the accumbens nucleus (Acb)and olfactory tubercle (OT).Antibodies raised against C-terminal portions of the prepropeptides labeled cell bodies of neurons with diameters of 8^15W m.PPD-,PPE-and PPTA-immunoreactive neurons were distributed throughout the Acb and concentrated in the dense cell layer of the OT.PPTB-immunoreactive neurons were observed to form cell clusters,which were localized in W -opioid receptor-immunoreactive patchy regions in the Acb,but were very rarely found in the dense cell layerof the OT.Double-immuno£uor escence analysis r evealed that PPD,PPE and PPTB immuno-reactivities were shown in 69%,19%and 14%of PPTA-immunoreactive neurons,respectively,in the Acb core region,and in 92%,7%and 25%of PPTA-immunoreactive neurons,respectively,in the Acb shell region.In the olfactory bulb,51%,19%and 3%of PPTA-immunoreactive neurons showed PPD,PPE and PPTB immunoreactivities,respectively.PPD and PPE immunoreactivities were rarely coexpressed in single neurons of all striatal regions.The present results indicated that,although PPTA and PPE were occasionally coexpressed in single neurons of the ventral striatum,the segregated expression of PPD and PPE in the ventral striatum was similar to that in the dorsal striatum.The clustered localization of PPTB-expressing neurons in the Acb and near absence of PPTB-expressing neurons in the dense cell layer of the OT suggests that neurokinin B is a key substance in di¡erentiating between the ventral and dorsal striatal regions.ß2002IBRO.Published by Elsevier Science Ltd.All rights reserved.Key words:ventral striatum,prepropeptides,medium-sized neuron,opioids,tachykinins,rat.The striatal regions,such as the neostriatum,accumbens nucleus (Acb)and olfactory tubercle (OT),share com-mon elemental properties,such as intense immunoreac-tivity for tyrosine hydroxylase,and very high expression of mRNAs encoding dopamine receptors (Meador-Woodru¡et al.,1989;Mengod et al.,1989;Mansour et al.,1991),preprodynorphin (PPD,Merchenthaler et al.,1997),preproenkephalin (PPE,Harlan et al.,1987)and preprotachykinin A (PPTA,Warden and Young,1988;Harlan et al.,1989).However,the striatal regions are considered to play di¡erent roles with respective ana-tomical connections:the neostriatum is involved in thecontrol of voluntary movements,receiving inputs mainly from the neocortex and sending output to the globus pallidus,entopeduncularnucleus and substantia nigr a (SN)(forr eview,see Wilson,1998);the Acb plays a role in adaptive behavioral responses (Mogenson et al.,1980;Le Moal and Simon,1991),receiving inputs mainly from limbic structures such as the hippocampus,prefrontal cortex and amygdala and sending output to the ventral pallidum (VP),ventral tegmental area (VTA)and SN (forr eview,see Groenewegen et al.,1999;Zahm,1999);the OT takes part in the olfactory system,receiv-ing inputs mainly from the olfactory bulb and olfactory cortex (Newman and Winans,1980;Luskin and Price,1983;Price,1985;Josephson et al.,1997).The vast majority of neurons in all the striatal regions are medium-sized spiny cells (Wilson and Groves,1980;Millhouse and Heimer,1984;Chang and Kitai,1985).These medium-sized spiny cells are known to be GABAergic projection neurons and express at least one of the peptide neurotransmitters,i.e.dynorphins,enke-phalins,substance P and neurokinin B (for review,see Gerfen,1992;Kawaguchi,1997).Dynorphins,enkepha-lins,substance P and neurokinin B are encoded in the mRNAs for PPD,PPE,PPTA and preprotachykinin B (PPTB),respectively (for review,see Ho «llt,1983;Nawa611*Correspondence to:T.Kaneko,Department of Morphological Brain Science,Graduate School of Medicine,Kyoto University,Kyoto 606-8501,Japan.Tel.:+81-75-753-4331;fax:+81-75-753-4340.E-mail address:kaneko@mbs.med.kyoto-u.ac.jp (T.Kaneko).Abbreviations:Acb,accumbens nucleus;AcbC,core region of the accumbens nucleus;AcbS,shell region of the accumbens nucleus;DARPP32,dopamine-and cAMP-regulated phosphoprotein of 32kDa;OT,olfactory tubercle;PPD,preprodynorphin;PPE,preproenkephalin;PPTA,preprotachykinin A;PPTB,prepro-tachykinin B;SN,substantia nigra;SNr,substantia nigra pars reticulata;VP,ventral pallidum;VTA,ventral tegmental area.Neuroscience Vol.114,No.3,pp.611^627,2002ß2002IBRO.Published by ElsevierScience LtdAll rights reserved.Printed in Great BritainPII:S 0306-4522(02)00312-30306-4522/02$22.00+0.00et al.,1983;Kawaguchi et al.,1986;Krause et al.,1987; Bonneret al.,1987).In the neostriatum,the expression pattern of these peptide neurotransmitter-encoding genes corresponds to classi¢cations of the neostriatal projec-tion neurons by their target areas:neostriatonigral and neostriato-entopeduncular neurons are characterized by co-production of PPTA and PPD;neostriatopallidal neurons are distinguished by expression of PPE(Gerfen and Young,1988;Lee et al.,1997).In addition,we have recently reported that PPTB-producing neurons send axons predominantly to the substantia innominata (Furuta et al.,2000).Since PPD,PPE,PPTA and PPTB are not expressed in neostriatal interneurons (Lee et al.,1997;Furuta et al.,2000)which are charac-terized by expression of chemical markers such as choline acetyltransferase,somatostatin,calretinin or parvalbu-min(forr eview,see Kawaguchi et al.,1995),these pre-propeptides are selective markers for projection neurons in the dorsal striatum.It is thus expected that PPD,PPE, PPTA and PPTB are projection neuron markers in the ventral striatum as well as in the dorsal striatum. Single expression or coexpression of PPD,PPE,PPTA and PPTB in projection neurons of the ventral striatum is not well known,although many ventral striatal neu-rons are known to express PPD,PPE and PPTA as introduced above.Recently,Lee et al.(1997)and Kaneko et al.(1998)produced antibodies to the C-ter-minal portion of PPD,PPE,PPTA and PPTB,and suc-ceeded in visualizing cell bodies of neurons expressing those prepropeptide without colchicine treatment.In the present study,we analyzed the expression of those prepropeptides in single neurons of the ventral striatum by double-£uorescence immunocytochemistry with the antibodies against C-terminal portions of the prepropep-tides.In addition,immunoreactivities for the markers of striatal interneurons and immunoreactivity for dopa-mine-and cAMP-regulated phosphoprotein of32kDa (DARPP32),which is known to be expressed in the vast majority of striatal medium-sized neurons(Ouimet et al.,1984;Walaas and Greengard,1984),were exam-ined in the ventral striatal neurons immunoreactive for PPD,PPE,PPTA and PPTB by the double-immuno£uo-rescence methods.EXPERIMENTAL PROCEDURESThe procedures of the present experiments were in accordance with the rules of animal care by the Institute of Laboratory Animals,Graduate School of Medicine,Kyoto University (Japan).Fixation and immunoperoxidase stainingNine Wistarr ats(200^250g body weight;Japan SLC,Shi-zuoka,Japan)were deeply anesthetized by i.p.injection of35 mg chloral hydrate/100g body weight,and perfused transcar-dially with200ml of5mM phosphate-bu¡ered0.9%(w/v)sa-line(PBS;pH7.4),followed by300ml of3%(w/v) formaldehyde in75%saturated picric acid and0.1M sodium phosphate(pH7.4).After¢xation,rat brain blocks were cryo-protected with30%(w/w)sucrose in PBS,and cut into20-or 30-W m-thick frontal sections on a freezing microtome.The sections were incubated overnight with1W g/ml a⁄nity-puri¢ed guinea-pig antibody raised against the C-terminal ico-sapeptide of PPD,nonadecapeptide of PPE,pentadecapeptide of PPTA or docosapeptide of PPTB.The production and char-acterization of the anti-prepropeptide antibodies were reported in ourpr evious studies(Lee et al.,1997;Kaneko et al.,1998). Prepropeptide immunoreactivity was not detected with the absorbed antibodies.The incubations were carried out at room temperature in PBS containing0.3%(v/v)Triton X-100, 0.25%(w/v)V-carrageenan,1%(v/v)donkey serum and0.02% (w/v)sodium azide(PBS-XCD),and followed by a rinse with PBS containing0.3%(v/v)Triton X-100(PBS-X).The sections were further incubated for1h with10W g/ml biotinylated anti-guinea-pig IgG donkey antibody(Jackson,West Grove,PA, USA)and then for1h with avidin-biotinylated per oxidase com-plex(ABC-Elite kit;Vector,Burlingame,CA,USA)in PBS-X. Finally,the bound peroxidase was developed by reaction with 0.02%(w/v)diaminobenzidine^4HCl and0.001%(v/v)H2O2in 50mM Tris^HCl,pH7.6.The sections were mounted onto gelatin-coated glass slides,dehydrated in ethanol series,cleared in xylene,and coverslipped.Some sections were treated with Cresyl Violet for Nissl counterstain after peroxidase reaction. Double-immuno£uorescence histochemistryAll the following incubations were carried out in PBS-XCD at room temperature.The brain sections were incubated overnight with a mixture of a guinea-pig antibody and either rabbit or mouse antibody.The primary antibodies were selected from the antibodies listed in Table1.Aftera r inse with PBS-X,the sections were incubated for1h with10W g/ml biotinylated anti-guinea-pig IgG oranti-mouse IgG donkey antibody(Jackson). Finally,the sections were incubated with a mixture of1W g/ml Alexa594-conjugated streptavidin(Molecular Probes,Eugene, OR,USA)and10W g/ml dichlorotriazinylamino£uorescein-con-jugated anti-rabbit IgG donkey antibody(Chemicon)in PBS-XCD containing10%(v/v)normal guinea-pig or mouse serum. The sections were mounted onto gelatin-coated glass slides,air-dried,and coverslipped with50%(v/v)glycerol and2.5%(w/v) triethylenediamine(anti-fading agent)in PBS.Data analysisImmuno£uorescence was observed under an epi£uorescence microscope,Axiophot(Zeiss,Oberkochen,Germany),with appropriate¢lter sets for£uorescein(excitation,450^490nm; emission,514^565nm)and Alexa594(excitation,530^585nm; emission,v615nm).Immunopositive neurons were counted under the epi£uorescence microscope in two to four double-im-munostained frontal sections through the Acb and OT for each staining combination.The sections were also observed under a confocal laser-scanning microscope(LSM410;Zeiss)by using an appropriate excitation laser beam and an emission¢lter for Alexa594(excitation,543nm;emission,v570nm)and£uo-rescein(excitation,488nm;emission,510^525nm),and cap-tured by using a software(LSM;Zeiss).When one of the primary antibodies was omitted or replaced with normal IgG or serum,no immuno£uorescence for the omitted or replaced antibody was detected.Numbers of neurons immunopositive for the prepropeptides, DARPP32or interneuron markers were counted under the epi£uorescence microscope in visual¢elds(40U objective)not overlapping one another,but covering most areas of the core or shell region of the Acb(AcbC and AcbS,respectively)or OT in the frontal sections.Three rats were used for double-immuno-£uorescence staining of each combination of the prepropeptides. At least two rats were used for double-immuno£uorescence staining for DARPP32or interneuron markers and the prepro-peptides,and sections from the rat showing the best staining were selected for counting.The data were statistically compared by M2test with2U3contingency tables between the fourstr iatal regions,AcbC,AcbS,OT and neostriatum.All the black and white photographs were printed in a usual way,and digitized with a scannerat300dpi r esolution.TheT.Furuta et al. 612digital images were arranged in software Canvas(Deneba Sys-tems,Miami,FL,USA)without contrast change,and saved as TIFF¢les.The delineation of cytoarchitectonic regions was determined according to Paxinos and Watson(1998).Further-more,the borders between the core and shell regions in the Acb were demarcated with calbindin-D28k immunoreactivity in adjacent sections(Zahm and Brog,1992;Jongen-Re“lo et al., 1994).Table1.Primary antibodies used in double-immuno£uorescence histochemistryAntibody ConcentrationGuinea-pig antibody to C-terminal icosapeptide of rat PPD11W g/mlGuinea-pig antibody to C-terminal nonadecapeptide of rat PPE11W g/mlRabbit antibody to C-terminal nonadecapeptide of rat PPE11W g/mlGuinea-pig antibody to C-terminal pentadecapeptide of rat PPTA11W g/mlRabbit antibody to C-terminal pentadecapeptide of rat PPTA11W g/mlRabbit antibody to C-terminal docosapeptide of rat PPTB21W g/mlGuinea-pig antibody to C-terminal docosapeptide of rat PPTB21W g/mlGuinea-pig antibody to W-opioid receptor31W g/mlMouse antibody to DARPP3241:20000Mouse anti-parvalbumin ascites(IgG)51:2000Rabbit anti-calretinin antiserum61:2000Rabbit anti-choline acetyltransferase antiserum61:2000Rabbit anti-somatostatin antiserum71:5000Rabbit anti-calbindin-D28k61W g/mlThe sources of antibodies are as follows:1Lee et al.,1997;2Kaneko et al.,1998;3Kaneko et al.,1995,4generous gift from Dr.Nishi;Depart-ment of Physiology,Kurume University(Ouimet et al.,1984);5Sigma,St.Louis,MO,USA;6Chemicon,Temecula,CA,USA;7Peninsula, Belmont,CA,USA.Fig.1.Distributions of immunoreactivities for PPD,PPE,PPTA and PPTB in the Acb.PPD,PPE and PPTA immunoreac-tivities were observed throughout the Acb,showing heterogeneous distributions in density.Arrows in a and c indicate intenseimmunoreactivity for PPD and PPTA,respectively,in the dorsomedial part of the shell region of the Acb.PPTB immunore-activity was located in a patch-like manner(arrows in d)and in the lateral stripe of the striatum(LSS).ac,anterior commis-sure;CPu,caudate^putamen;VP,ventral pallidum.The calibration bar is applied to all the photographs.Expression pattern for prepropeptides in the ventral striatum613T.Furuta et al. 614Fig.2.RESULTSDistributions of prepropeptide immunoreactivities in the Acb and OTPPD,PPE and PPTA immunoreactivities were observed throughout the AcbC and AcbS,though these immunoreactivities were not distributed homogeneously.PPD immunoreactivity was intense in a patch-like man-ner(Fig.1a )and especially in the dorsomedial portion of the AcbS (arrow in Fig.1a )where intense PPTA immu-noreactivity was also found (arrow in Fig.1c ).PPE im-munoreactivity was weaker in the AcbS than in the AcbC (Fig.1b ).PPTB immunoreactivity was distributed in a patch-like manner (arrows in Fig.1d ),while PPD,PPE and PPTA immunoreactivities were distributedthroughout the Acb.In addition,PPTB immunoreactiv-ity was intense in the lateral stripe of the striatum.In the OT,PPD,PPE and PPTA immunoreactivities were densely distributed in the dense cell layer,and relatively weak immunoreactivities were observed in the multiform layer(Fig.2a^c ).It was interesting that only a little PPTB immunoreactivity was observed in the OT (Fig.2d ).Immunoreactivities for PPD,PPE,PPTA and PPTB were observed mainly in neuronal cell bodies with diam-eters of 8^15W m in the Acb and OT (Fig.3).The immu-noreaction products appeared to be associated with some membraneous structures in the perikarya similarly to those of the neostriatum.In the counterstained sections,PPD,PPE,PPTA and PPTB immunoreactivities were shown in 47.1%(1139/2417),29.0%(710/2450),61.4%Fig.2.Distributions of immunoreactivities for PPD,PPE,PPTA and PPTB in the OT.PPD,PPE and PPTA immunoreactiv-ities were dense in the dense cell layer,and weak in the multiform layer (a^c).Very little PPTB immunoreactivity was found in the OT.Arrows indicate the ventral convolution (‘cap-regions’)of the dense cell layer where PPD,PPTA and PPTB immunoreactivities were observed but PPE immunoreactivity was absent.An open arrow in c indicates neuropil showing im-munoreactivity for PPTA in the multiform layer associated with the ‘cap-regions’.A double arrow in d indicates PPTB-im-munoreactive cell cluster in the multiform layer of the OT.VP,ventral pallidum.The calibration bar is applied to all thephotographs.Fig.3.PPD-,PPE-,PPTA-and PPTB-immunoreactive cell bodies in the Acb (a^d)and OT (e^g).PPD,PPE,PPTA and PPTB immunoreactivities were observed in medium-sized neurons with diameters of 8^15W m (measured in 50neurons for each prepropeptide).No signi¢cant di¡erence in size between PPD-,PPE-,PPTA-and PPTB-immunoreactive neurons was noticed.Almost no PPTB-immunopositive neurons were found in the dense cell layer of the OT (h).The calibration bar isapplied to all the photographs.Expression pattern for prepropeptides in the ventral striatum 615(1463/2384)and 11.0%(294/2684),respectively,of neu-rons in the Acb,and 44.8%(602/1343),49.2%(586/1192),62.0%(623/1005)and 1.3%(18/1348),respectively,of neurons of the OT.Furthermore,in the counterstained sections which were immunostained with a mixture of primary antibodies for PPD,PPE,PPTA and PPTB,the vast majority of medium-sized neurons in the Acb and OT showed immunoreactivity for at least one of those prepropeptides.PPD-,PPE-or PPTA-immunore-active neurons were distributed throughout the Acb and densely packed in the dense cell layerof the OT (Fig.4).It should be noted that the distribution of PPD-immu-noreactive neurons was not so patchy as that of PPD immunoreactivity (Fig.1a ),since many weakly PPD-pos-itive neurons were distributed in the weakly PPD-immu-noreactive regions.Although no rostrocaudal di¡erence in occurrence of immunoreactive neurons was noticed,distributions of these neurons were sparser in the inter-mediate part of the AcbS than in the medial and lateral parts of the AcbS.In addition,PPD-and PPTA-immu-noreactive neurons were frequently found in the dorso-medial part of the AcbS (arrows in Fig.1a,c ).In the islands of Calleja which are composed of densely packed granule cells,mRNA encoding PPTA has been reported to be expressed (Warden and Young,1988;Harlan et al.,1989).In the sections stained forPPTA,we obser vedsmall punctate immunoreactivity for PPTA in the islandsof Calleja.This PPTA immunoreactivity did not show clearimages of cell body,but it was localized in the cell bodies in the counterstained sections.No PPD,PPE or PPTB immunoreactivity was observed in the islands of Calleja.In the OT,almost no neurons showed PPTB immunoreactivity in the dense cell layer and molecular layer,although a few PPTB-immunoreactive neurons were distributed in the multiform layer (double arrow in Fig.2d ).Colocalizations of immunoreactivities for PPD,PPE,PPTA and PPTB in single neurons of the Acb and OT We next investigated how immunoreactivities for PPD,PPE,PPTA and PPTB were colocalized in single neurons of the Acb and OT,using the double-immuno£uores-cence method (Tables 2and 3,Figs.5^7).Using three rats,we obtained the numbers of neurons that were immunopositive for the prepropeptides.The data from the three rats were added up and presented in Table 2,whereas the percentages of colocalization were averaged among the three rats in Table 3.Coexpression percen-tages of PPD,PPE and PPTA immunoreactivities in PPTB-immunoreactive neurons were noticed to be the most variable of thedata.Fig. 4.Distribution of PPD-,PPE-,PPTA-and PPTB-immunoreactive neurons in the ventral striatum.Each ¢lled circle represents 10immunoreactive neuronal cell bodies in a,b and c.Arrows in b indicate the ventral convolutions of dense cell layer where PPE-immunoreactive neuron was not found.In d,each small ¢lled circle represents a PPTB-immunoreactive neuronal cell body.ac,anterior commissure;AcbC,core region of the Acb;AcbS,shell region of the Acb;CPu,caudate^putamen;LSS,lateral stripe of the striatum;OT,olfactory tubercle;VP,ventral pallidum.The calibration bar is applied toall the photographs.T.Furuta et al.616In the AcbC,only a few PPE-immunoreactive neurons showed PPD (2%)orPPTB immunor eactivities (2%),whereas a considerable number of PPE-immunoreactive neurons displayed PPTA immunoreactivity (27^32%).PPTA immunoreactivity was observed in the vast major-ity of PPD-(94%)or PPTB-immunoreactive neurons (82^83%).Furthermore,79^80%of PPTB-immunoreac-tive AcbC neurons were immunopositive for PPD.AcbS neurons showed mostly the same results of co-localization of prepropeptide immunoreactivities as AcbC neurons except that PPTB-immunoreactive neu-rons in the AcbS were less frequently immunoreactive forPPTA (47^50%)and PPD (61^62%),and that PPE-immunoreactive neurons were less frequently immunore-active forPPTA (13^15%)than those in the AcbC.In the OT,immunoreactivities for PPD and PPE were rarely colocalized in single neurons (2^3%)similarly to those in the neostriatum and Acb.More than 90%of PPD-immunoreactive neurons and 21^22%of PPE-immunopositive neurons showed PPTA immunoreactivi-ty.About half or18^19%of PPTA-immunoreactive neu-rons were immunoreactive for PPD or PPE,respectively.PPTB-immunoreactive neurons that were observed only in the multiform layer of the OT frequently coexpressed PPD (67^71%)orPPTA (76^80%),but did not show PPE immunoreactivity.Table 3.Mean percentages of colocalization of prepropeptide immunoreactivity in medium-sized neurons of the ventral striatumSitesNeurons positive for:Coexisting prepropeptide (numerator)(denominator)PPTA PPD PPE PPTB Accumbens PPTA ^73.6þ3.9%a 13.0þ5.6%15.1þ0.9%Core regionPPD 94.4þ2.6%^1.1þ0.4%18.2þ1.7%PPE 26.9þ11.7% 1.9þ0.7%^1.9þ1.0%PPTB 82.5þ2.3%78.7þ10.0% 5.0þ2.4%^Accumbens PPTA ^88.0þ3.4% 6.1þ1.0%22.9þ3.6%Shell regionPPD 84.2þ4.4%^3.0þ0.7%14.5þ2.7%PPE 14.5þ3.9% 6.4þ2.3%^2.4þ1.3%PPTB 46.7þ15.8%60.9þ11.5% 6.7þ3.6%^Olfactory tuberclePPTA ^51.5þ4.9%18.3þ1.2% 4.0þ2.3%PPD 93.2þ2.2%^2.8þ0.9% 5.7þ3.7%PPE 20.5þ2.3% 2.1þ0.9%^0%PPTB75.6þ23.0%66.9þ15.3%0%^Three rats were used for double-immuno£uorescence staining of each combination of the prepropeptides.The counting was done in every rat as that in Table 2and percentages of colocalization of the prepropeptides in single neurons were calculated in each rat.aMean þS.D.Table 2.Numbers of neurons coexpressing two of the prepropeptides in the dorsal and ventral striatumSitesNeurons positive for:Coexisting prepropeptide (numerator)(denominator)PPTAPPDPPEPPTB Accumbens PPTA ^657/908(72.4%)111/725(15.3)142/952(14.9)Core regionPPD 657/702(93.6)*2#^6/570(1.1)168/914(18.4)PPE 111/352(31.5)*2#6/354(1.7)2#^9/449(2.0)PPTB 142/173(82.1)*2#168/211(79.6)2#9/171(5.3)2#^Accumbens PPTA ^535/6.4(88.6)29/473(6.1)116/492(23.6)Shell regionPPD 535/642(83.3)2#^15/503(3.0)82/558(14.7)PPE 29/229(12.7)2#15/281(5.3)2#^11/455(2.4)PPTB 116/233(49.8)2#82/132(62.1)2#11/157(7.0)2#^Olfactory PPTA ^501/971(51.6)139/733(19.0)36/976(3.7)TuberclePPD 501/540(92.8)#^10/415(2.4)27/512(5.3)PPE 139/632(22.0)#10/612(1.6)#^0/651(0.0)PPTB 36/45(80.0)#27/38(71.1)#0/29(0.0)#^Neostriatum aPPTA ^1578/1657(95.2)28/1479(1.9)109/1613(6.8)PPD 1578/1645(95.9)^4/1570(0.3)42/1031(4.1)PPE 28/1594(1.8)4/1830(0.2)^41/1010(4.1)PPTB109/167(65.3)42/166(25.3)41/137(29.9)^For each double-immunostaining,striatal neurons were counted under the epi£uorescence microscope in visual ¢elds (40U objective)not over-lapping one another,but covering most areas of the OT and core and shell region of the Acb in three frontal sections from every rat.The data from three rats were added up to the total numbers for each combination.Asterisks (*),crosses (2)and sharps (.)indicate that the expression pattern for the two prepropeptides in the region is signi¢cantly di¡erent (P 60.001;M 2test)from that in the shell region,OT and neostriatum,respectively.For further detail,see Results .aThe data of the neostriatum were obtained from the results of Lee et al.(1997)and Furuta et al.(2000)forcompar ison.Expression pattern for prepropeptides in the ventral striatum617The expression patterns for each combination of the prepropeptides were statistically compared by M 2test with 2U 3contingency tables between the fourstr iatal regions (Table 2).The expression pattern for PPE/PPTB was not signi¢cantly di¡erent (P s 0.05)between the AcbC and AcbS.The di¡erence in the expression pattern for PPD/PPE was weakly signi¢cant between the AcbC and AcbS (0.016P 60.05).The di¡erence in the expression pattern for PPD/PPTB was signi¢cant between the AcbC and AcbS (0.0016P 60.01).All the other expression patterns showed highly signi¢cant dif-ferences (P 60.001)between the four striatal regions.PPTA-immunoreactive neurons in the islands of Calleja were not counted in the double-staining study because the immunoreactivity was not shown with a clear image of single cell bodies.However,no PPTA immu-noreactivity was thought to be colocalized with PPD,PPE or PPTB immunoreactivity in single neurons ofthe islands of Calleja,since neurons in the islands of Calleja did not show immunoreactivity for PPD,PPE orPPTB.Colocalization of PPD,PPE,PPTA and PPTB immunoreactivities with immunoreactivities for DARPP32and striatal interneuron markersWe further investigated immunoreactivity for DARPP32in the PPD-,PPE-,PPTA-orPPTB-immu-noreactive neurons of the ventral striatum by double-im-munostaining (Table 4).DARPP32immunoreactivity was observed in 74%,85%,81%and 47%of medium-sized neurons immunoreactive for PPD,PPE,PPTA and PPTB,respectively,in the AcbC.In the AcbS,80%of PPD-immunoreactive neurons,77%of PPE-im-munoreactive neurons,82%of PPTA-immunoreactive neurons and 22%of PPTB-immunoreactiveneuronsFig.5.Double-immuno£uorescence staining of PPTA and PPE (a,a P ),PPTA and PPD (b,b P )and PPD and PPE (c,c P ,c Q )in the Acb.Photomicrographs in each row were taken from the same site under di¡erent excitations:a^c for £uorescein;a P ,b P and c Q forAlexa594.c P was taken by double exposure.Arrows indicate PPTA-immunoreactive neurons coexpressing PPE (a,a P )orPPD (b,b P ).Arrowheads indicate single-labeled neurons.The calibration bar is applied to all the photographs.T.Furuta et al.618showed DARPP32immunoreactivity.The vast majority of PPD-(93%),PPE-(95%)and PPTA-(89%)immuno-reactive neurons were immunopositive for DARPP32in the OT.When the sections were doubly stained with one of the antibodies for striatal interneuron markers and the mix-ture of the guinea-pig antibodies to PPD,PPE,PPTA and PPTB,no neurons immunoreactive for the prepro-peptides showed immunoreactivity for the interneuron markers in the AcbC,AcbS or OT (Table 5).Thus,PPD,PPE,PPTA and PPTB were suggested to be spe-ci¢cally expressed in projection neurons.Table 4.DARPP32immunoreactivity in PPD-,PPE-,PPTA-and PPTB-producing neuronsNeurons positive for (denominator)DARPP32-immunopositive neurons (%)Accumbens Accumbens Olfactory tubercle Core regionShell region PPD 142/192(74.0)136/170(80.0)98/106(92.5)PPE 158/186(84.9)85/111(76.7)101/106(95.3)PPTA 117/144(81.3)95/116(81.9)109/122(89.3)PPTB33/70(47.1)17/78(21.8)^For each double-immunostaining,striatal neurons were counted under the epi£uorescence microscope in visual ¢elds not overlapping one another and covering almost the whole area of the core or shell region of the Acb or OT in the four 20-W m-thick frontalsections.Fig.6.Double-immuno£uorescence staining of PPTB and PPTA (a,a P ),PPTB and PPD (b,b P )and PPTB and PPE (c,c P ,c Q )in the Acb.Photomicrographs in each row were taken from the same site under di¡erent excitations:a^c for £uorescein;a P ,b P and c Q forAlexa594.c P was taken by double exposure.Arrows indicate PPTB-immunoreactive neurons coexpressing PPTA (a,a P )orPPD (b,b P ).Arrowheads in c,c P and c Q indicate PPTB-immunoreactive neurons not showing PPE immuno-reactivity.The calibration bar is applied to all the photographs.Expression pattern for prepropeptides in the ventral striatum 619。