Subcellular Distribution of5-Hydroxytryptamine2A and

N-Methyl-D-Aspartate ReceptorsWithin Single Neurons in Rat Motor

and Limbic Striatum

J.J. RODRIGUEZ, D.R. GARCIA, AND V.M. PICKEL*Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College

of Cornell University, New York, New York 10021

ABSTRACTThe dorsolateral caudate-putamen nucleus (CPN) and the nucleus accumbens (NAc)

shell, respectively, are involved in many motor and limbic functions that are affected byactivation of the 5-hydroxytryptamine2A receptor (5HT2AR) and the N-methyl-D-aspartatesubtype of glutamate receptor (NMDAR). We examined the functional sites for 5HT2ARactivation and potential interactions involving the NMDAR subunit NR1 (NMDAR1) withinthese striatal regions. For this examination, sequence-specific antipeptide antisera againstthese receptors were localized by electron microscopic dual-labeling immunocytochemistry inthe rat brain. In the dorsolateral CPN and the NAc shell, the 5HT2AR-labeled profiles weremainly dendrites, but somata and axons were also immunoreactive. The neuronal somatacontained round unindented nuclei that are typical of spiny striatal neurons, although fewdendritic spines were 5HT2AR immunolabeled. In all neuronal profiles, the 5HT2AR labelingwas primarily associated with cytoplasmic organelles and more rarely was localized tosynaptic or nonsynaptic plasma membranes. Colocalization of 5HT2AR and NMDAR1 wasseen primarily in somata and dendrites. Significantly greater numbers of 5HT2AR- or 5HT2AR-and NMDAR1-containing dendrites were seen in the dorsolateral CPN than in the NAc shell.As compared with 5HT2AR, NMDAR1 labeling was more often observed in dendritic spines,and these were also more numerous in the CPN. These results indicate that 5HT2A and NMDAreceptors are coexpressed but differentially targeted in single spiny striatal neurons and arelikely to play a major role in control of motor functions involving the dorsolateral CPN. J.Comp. Neurol. 413:219–231, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: serotonin; glutamate; synaptic plasticity; atypical antipsychotic; caudate-putamen

nucleus; nucleus accumbens

Fourteen serotonin (5-hydroxytryptamine, 5HT) recep-tor subtypes have been identified and divided into sevenfamilies (5HT1–7) according to their structural, transduc-tional, and functional properties (Hoyer et al., 1994; Hoyerand Martin, 1997). 5HT2A receptor (5HT2AR) binding sitesand mRNA are distributed in regions throughout thebrain, including the caudate-putamen (CPN) and nucleusaccumbens (NAc; Pazos et al., 1985; Pompeiano et al.,1994; Mijnster et al., 1997). This regional localization of5HT2AR has also been shown by light microscopic immuno-cytochemical localization of antisera that were raisedagainst peptide sequences within the cloned receptor(Morilak et al., 1993, 1994; Hamada et al., 1998; Wu et al.,1998).

The presence of 5HT2AR and the known serotonergicinnervation of the NAc shell and CPN (Soghomonian et al.,1987, 1989; Van Bockstaele and Pickel, 1993; Van Bocks-taele et al., 1996) suggest involvement in limbic and motorfunctions, respectively, that are ascribed to these regions

Grant sponsor: National Institute on Drug Abuse; Grant number:DA04600; Grant sponsor: National Institute of Mental Health; Grantnumbers: MH40342 and MH00078.

*Correspondence to: Virginia M. Pickel, Department of Neurology andNeuroscience, Division of Neurobiology, Weill Medical College of CornellUniversity, 411 East 69th Street, New York, NY 10021.E-mail: vpickel@mail.med.cornell.edu

Received 16 February 1999; Revised 22 June 1999; Accepted 23 June1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 413:219–231 (1999)

r 1999 WILEY-LISS, INC.

(Welsh et al., 1998). As the target for converging excitatoryafferents from the prefrontal cortex, hippocampus, andamygdala (Lopes da Silva et al., 1984; Mogenson andNielsen, 1984; Yang and Mogenson, 1984; Christie et al.,1987; Groenewegen et al., 1987; McGeorge and Faull,1989; Mulder et al., 1998), the NAc shell region is ideallysuited for integration of locomotor activity with cognitivememory functions and associative learning (Welsh et al.,1998). In contrast, the CPN receives major glutamatergicinput from the somatosensory cortex and is critical formotor function (McGeorge and Faull, 1989).

Glutamatergic excitation of striatal neurons is mediatedin part through activation of N-methyl-D-aspartate recep-tors (NMDARs) that are located mainly at postsynapticsites in dendrites and dendritic spines (Gracy and Pickel,1996; Gracy et al., 1999). The prominent distribution indendritic spines is consistent with involvement of NMDARin synaptic plasticity because spines are subject to dra-matic changes with environmental manipulations, includ-ing chronic treatment with haloperidol and other typicalantipsychotic drugs (Kelley et al., 1997; Rodrıguez andPickel, 1999). Involvement in synaptic plasticity is a rolethat may be shared by 5HT2AR because 5HT2AR antago-nists prevent or reverse phencyclidine-induced psychosisattributed to blockade of NMDAR (Wang and Liang, 1998).In addition, both 5HT2AR and NMDAR have been impli-cated in synaptic changes during development (Hofer andConstantine-Paton, 1994; Brooks et al., 1997; Choi et al.,1997; Das et al., 1998) and following repetitive glutamater-gic excitation within the CPN of rats and humans (Hornyk-iewick, 1993; Anglade et al., 1996; Ingham et al., 1998). Itis not known, however, whether dendritic spines areenriched in 5HT2AR or whether the same neurons express5HT2AR and NMDAR in either the dorsolateral CPN or theNAc shell. To address these questions, we examined theelectron microscopic immunocytochemical localization of5HT2AR and the NMDAR R1 subunit (NMDAR1), which isa major subunit essential for ionophore function (Mon-aghan et al., 1989; Hollman and Heinemann, 1994), in thedorsolateral CPN and the NAc shell of rat brain.

MATERIALS AND METHODS

Tissue preparation

The animal protocols used in the present study adhere tothe NIH guidelines for the Care and Use of LaboratoryAnimals in Research and were approved by the AnimalCare Committee at Weill Medical College of Cornell Univer-sity. Adult male Sprague-Dawley rats (450–550 g; Taconic,Germantown, NY) were deeply anestethized with sodiumpentobarbital (100 mg/kg, i.p.). The brains of these ani-mals were fixed by aortic arch perfusion with 50 ml of 3.8%acrolein (Polysciences, Warrington, PA) in a solution of 2%paraformaldehyde and 0.1 M phosphate buffer (PB), pH7.4, followed by 250 ml of 2% paraformaldehyde. Thebrains were removed from the cranium and cut into4–5-mm coronal slabs of tissue containing the entirerostrocaudal extent of the CPN and NAc. This tissue wasthen postfixed for 30 minutes in 2% paraformaldehyde andsectioned at 30–40 µm on a Vibratome (Leica, Inc., Deer-field, IL; Leranth and Pickel, 1989). To remove the excessof reactive aldehydes, sections were treated with 1%sodium borohydride in 0.1 M PB for 30 minutes. The tissuesections were then freeze–thawed to optimize the penetra-tion of immunoreagents. For this procedure, sections were

(1) incubated in cryoprotectant solution containing 25%sucrose and 3.5% glycerol in 0.05 M PB at pH 7.4 and (2)rapidly immersed in chloriduofluoromethane followed byliquid nitrogen and room-temperature PB. Sections werethen rinsed in 0.1 M PB and then in 0.1 M Tris-bufferedsaline (TBS), pH 7.6.

Antibodies

A monoclonal mouse antiserum was generated by usinga recombinant fusion protein between glutathione S-transferase and human 5HT2AR N-terminus (amino acids1–72) as the immunogen (Pharmingen, San Diego, CA). InWestern blot analysis, this 5HT2AR antiserum recognizeda band of 55 kD, corresponding to the predicted size of5HT2AR from rat brain lysates and a 5HT2AR-transfectedcell line (Wu et al., 1998). The immunolabeling with5HT2AR antiserum was also selectively removed by adsorp-tion with recombinant 5HT2AR but not with recombinant5HT2BR or 5HT2CR (Wu et al., 1998). In addition, recentreports have shown the specificity of 5HT2AR immunolabel-ing with this antiserum in primate cerebral cortex and ratskin (Carlton and Coggeshall, 1997; Jakab and Goldman-Rakic, 1998).

A polyclonal rabbit antiserum directed against a syn-thetic peptide corresponding to the C-terminus (aminoacids 909–938) of NMDAR1 was obtained from Chemicom(Temecula, CA). This antiserum has been shown specifi-cally to recognize four of the seven common splice variantsof the NMDA receptor: NR1–1a, NR1–1b, NR1–2a, andNR1–2b (Petralia et al., 1994, 1997; Gracy and Pickel,1996). Western blot analysis has also shown that the NR1antiserum produces a single major immunoreactive bandat Mr 5 120,000, the position expected for the amino acidsequence of NMDAR1 within the rat central nervoussystem (Petralia et al., 1994).

Dual immunocytochemical labeling

Tissue sections through the CPN and the NAc wereprocessed for combined immunoperoxidase and immuno-gold-silver labeling of 5HT2AR and NMDAR1 prior toplastic embedding. For this procedure, the Vibratomesections were first incubated for 30 minutes in 0.5% bovineserum albumin (BSA) in TBS to minimize nonspecificlabeling. The tissue sections then were incubated for 48hours at 4°C in 0.1% BSA in TBS containing (1) mousemonoclonal antiserum for 5HT2AR (1:1,000 dilution forimmunoperoxidase, 1:100 dilution for immunogold) and(2) rabbit polyclonal antiserum for NMDAR1 (1:250 dilu-tion for immunoperoxidase, 1:50 dilution for immunogold).

For immunoperoxidase labeling, sections were thenwashed and placed in (1) 1:400 dilution of either biotinyl-ated horse anti-mouse (for 5HT2AR) or goat anti-rabbit (forNMDAR1) immunoglobulin (IgG) and (2) 1:200 dilution ofbiotin-avidin complex from the Elite kit (Vector Laborato-ries, Burlingame, CA). All antisera dilutions were pre-pared in TBS, and the incubations were carried out atroom temperature. The peroxidase reaction product wasvisualized by incubation in a solution containing 0.022% of3,38 diaminobenzidine (Aldrich, Milwaukee, WI) and0.003% H2O2 in TBS for 6 minutes.

For immunogold-silver labeling, we used a modificationof the method described by Chan et al. (1990). Sectionswere rinsed in 0.1 M phosphate buffered saline, pH 7.4(PBS) and blocked in 0.8% BSA and 0.1% gelatin in PBSfor 10 minutes. Following this incubation, sections were

220 J.J. RODRIGUEZ ET AL.

processed for 2 hours in a 1:50 dilution of either goatanti-mouse (for 5HT2AR) or goat anti-rabbit (for NMDAR1)IgG conjugated with 1 nm colloidal gold (Amersham,Arlington, Heights, IL) in BSA/gelatin and then rinsed inBSA/gelatin and PBS. The bound gold particles weresecured in the tissue by placing the sections in 2% glutaral-dehyde in 0.01 M PBS for 10 minutes. These sections werethen washed in 0.2 M citrate buffer and reacted for 6–8minutes with a silver enhancement solution (IntenSE kit,Amersham). The silver reaction was stopped by successiverinses in citrate buffer.

Electron microscopicexamination and nomenclature

For electron microscopy, immunolabeled tissue sectionswere rinsed in 0.1 M PB, pH 7.4, and then postfixed for1 hour in 2% osmium tetroxide in PB. They were subse-quently dehydrated through a graded series of ethanolsand propylene oxide prior to embedding in Epon 812between sheets of Aclar plastic (Leranth and Pickel, 1989).Ultrathin sections were cut with a diamond knife (Dia-tome, Fort Washington, PA) and collected on copper meshgrids. The sections on grids were counterstained withuranyl acetate and lead citrate (Reynolds, 1963) andexamined with a Philips CM-10 electron microscope (Phil-ips, Mahwah, NJ).

Labeled profiles were classified as somata, dendrites,dendritic spines, unmyelinated axons, axon terminals, andglia according to the morphological features as defined byPeters et al. (1991). Somatic profiles were identified by thepresence of nuclei and abundant cytoplasm. Dendriteswere recognized by the presence of afferent axon terminalsand/or content of endoplasmic reticulum. The dendriticprofiles were differentiated from dendritic spines on thebasis of their larger size and abundance of mitochondriaand other organelles. As compared with spines, dendritesalso less frequently received input from axon terminalsforming asymmetric excitatory-type synapses. Dendriticspine heads were usually larger than small axonal or glialprofiles and had a more rounded bulbous shape. Unmyelin-ated axons were smaller than 0.2 µm in diameter andcontained microtubules and a few small synaptic vesicles(SSVs). Although unmyelinated axonal profiles were easilyrecognized in bundles, individual axons were sometimesdifficult to distinguish from spine necks. In this case, theywere included in the category of unidentified small pro-cesses. We defined axon terminals as profiles rangingbetween 0.2 and 1.5 µm in diameter and containing manySSVs. Synapses were defined as symmetric when havingthin pre- and postsynaptic densities or as asymmetricwhen having a thin pre- and a thick postsynaptic mem-brane specialization. Perforated synapses were defined asthose asymmetric synapses with a notable discontinuity(.50 nm) in the electron density of the postsynapticjunction (Greenough et al., 1978). Glial profiles werecharacterized by their irregular shape and investment ofneighboring neurons. In some cases, intermediate fila-ments were also used to identify astrocytic profiles.

Area density

To determine the area density (Sv, number/µm2) oflabeled profiles, the analysis was exclusively carried out onthe most superficial portions of the tissue in contact withthe embedding plastic to minimize artificial differences inlabeling attributed to potential differences in penetration

of reagents (Pickel et al., 1992). Regions used for thisanalysis were chosen based on the presence of 5HT2Aand/or NMDAR1 immunoreactivity and the morphologicalintegrity of the tissue. The labeled profiles were examinedin 12 Vibratome sections that were taken through the CPNand the NAc shell at a level 0.7–1.7mm anterior toBregma, according to the rat brain atlas of Paxinos andWatson (1986). The CPN and NAc shell were sampled fromthe same Vibratome sections. An area of 9,491 µm2 wasexamined in each randomly collected image, for a totalsampled area of 1,898.2 µm2 per animal and structure anda total of 5,694.6 µm2 per group of three animals in eachstructure.

Statistical analysis and figure preparation

Analysis of variance tests were used to examine differ-ences between the CPN and the shell of the NAc in terms ofnumbers of immunoreactive profiles and the number andtype of afferent inputs (implemented through Statview 4.1,Abacus Concepts). Figures were generated with Illustrator6.0 and Photoshop 4.0 (Adobe Inc.) and assembled withQuarkXpress 3.0 (Quark Inc.).

RESULTS

In the dorsolateral CPN, light microscopy showed5HT2AR-like immunoreactivity (5HT2AR-LI) within thickprocesses emanating from more lightly labeled somata(Fig. 1A). A similar labeling pattern was seen in the NAcshell (Fig. 1B), although the labeled processes were moreevident than somata in this region as compared with thedorsolateral CPN. The immunoreactive processes werepresumably dendrites because ultrastructural analysisshowed that, of the total 5HT2AR-labeled processes, 73.1%(285/390) in the dorsolateral CPN and 67.3% (228/339) inthe NAc shell were dendrites. In contrast, fewer than 6% ofthe labeled profiles in either region were dendritic spines,and the remainder was mainly axons and axon terminals.

NMDAR1-like immunoreactivity (NMDAR1-LI) was alsoseen by light microscopy as a peroxidase reaction productin somata and putative dendritic processes in the dorsolat-eral CPN and the NAc shell. Furthermore, in sectionsprocessed for dual labeling, the NMDAR1-immunoreactiveprocesses emanated from labeled somata, and many ofthese processes contained 5HT2A receptors in the dorsolat-eral CPN (Fig. 1C) and in the NAc shell (Fig. 1D). Electronmicroscopy showed somatic and dendritic localization ofNMDAR1 but also indicated that NMDAR1 was present inmany dendritic spines. Of the total NMDAR1-labeledprofiles, 40.3% (209/519) in the dorsolateral CPN and42.8% (199/444) in the NAc shell were dendrites, whereas29.3% (152/519) and 20.3% (90/444) were dendritic spinesin the respective striatal compartments.

Somatic 5-HT2AR distributionand relation with NMDAR1

In the dorsolateral CPN and NAc shell, many somatawere lightly labeled for 5HT2AR by using either the immu-noperoxidase or immunogold technique. These somatausually contained round nonindented nuclei that are typi-cal of spiny striatal projection neurons (DiFiglia et al.,1980; Figs. 2, 3). The peroxidase reaction product wasdistributed throughout the cytoplasm but appeared moreprevalent along membranes of smooth endoplasmic reticu-lum (SER), trans-Golgi membranes, and subsurface lamel-

STRIATAL 5HT2A AND NMDAR1 RECEPTORS 221

lae (Figs. 2, 2A, 3). Somata received sparse synaptic inputmainly from unlabeled terminals, and the postsynapticmembrane specializations rarely showed 5HT2AR immuno-reactivity.

In sections processed for dual labeling, almost all5HT2AR-immunoreactive somata contained NMDAR1-LI.

NMDAR1-LI was also seen mainly in the cytoplasm, wherethe labeling was associated with the tubulovesicular organ-elles including SER and Golgi lamellae (Figs. 2, 3). Occa-sionally, NMDAR1-LI was also seen along segments of theplasma and nuclear membranes in spiny neurons thatcontained cytoplasmic labeling (Figs. 2, 2A, 3).

Fig. 1. Brightfield photomicrographs showing striatal immunoper-oxidase localization of 5-hydroxytryptamine2A receptor (5HT2AR; A,B)and combined immunogold labeling for 5HT2AR and immunoperoxi-dase localization of N-methyl-D-aspartate subtype 1 of glutamatereceptor (NMDAR1) dual labeling (C,D). A,C: In the dorsolateralcaudate-putamen nucleus (CPN), 5HT2AR labeling is seen in thickprocesses (arrows) emanating from more lightly labeled somata

(asterisks). B,D: In the nucleus accumbens (NAc) shell, intense5HT2AR-like immunoreactivity is present in many thick processes(arrows), some of which appear contiguous with labeled somata(asterisks). In both the dorsolateral CPN and the NAc shell, NMDAR1immunoperoxidase labeling is seen in somata (asterisks) and proximalprocesses (arrows), many of which contain immunogold-silver labelingfor 5HT2AR (C,D). Scale bar 5 20 µm.

222 J.J. RODRIGUEZ ET AL.

Fig. 2. Electron micrographs from the dorsolateral caudate-putamen nucleus (CPN) showing the presence of 5-hydroxytrypt-amine2A receptor (5HT2AR) and N-methyl-D-aspartate subtype 1 ofglutamate receptor (NMDAR1) within a neuronal perikaryon andaxon initial segment. Immunogold-silver particles show NMDAR1labeling (arrows) throughout the cytoplasm. The NMDAR1 goldlabeling is occasionally located near the Golgi (G), along the plasmamembrane and nearby membranes of smooth endoplasmic reticulum(SER) and on or near the nuclear envelope of the nucleus (Nu).Immunoperoxidase labeling for 5HT2AR is seen as black precipitates(open arrowheads) that appear as cytoplasmic aggregates. The reac-tion product is also distributed along membranes of SER. The dual-labeled soma receives synaptic input from mainly unlabeled terminals

(UT). The boxed regions show portions of the soma that appose a5HT2AR-labeled axon (A) and the axon initial segment (B). A: 5HT2AR-labeled unmyelinated axon (5HT2A Ax) is apposed to the dual labeledsoma. Within the soma, immunoperoxidase reaction product for5HT2AR is seen along membranes of subsurface cisternae (SS), SER,and nearby mitochondria (M). The soma is apposed by a 5HT2AR-labeled glial process (5HT2A G). Two gold particles (arrows) identifyNMDAR1 on the inner leaflet of the nuclear membrane of nucleus(Nu). B: Initial axon segment shows immunogold NMDAR1 andimmunoperoxidase 5HT2AR labeling associated mainly with microtu-bules (MT). The axon receives an asymmetric (curved arrow) and asymmetric (arrowhead) synapse from unlabeled terminals (UT). Scalebars 5 1 µm, 0.4 µm in A,B.

Dendritic 5HT2AR distributionand relation with NMDAR1

5HT2AR was most prominently localized in dendrites,and these were seen in significantly higher numbers perunit area in the dorsolateral CPN than in the NAc shell(Fig. 4A). Dendrites were also the major profiles thatcontained NMDAR1 or NMDAR1 and 5HT2AR labeling(Fig. 4B,C). Whereas dendrites containing exclusivelyNMDAR1 showed no regional variations in number (Fig.4B), the dually labeled dendrites, like those containing5HT2AR, were detected in greater numbers in the dorsolat-eral CPN than in the NAc shell (Fig. 4C).

Most 5HT2AR-labeled dendrites were medium in diam-eter and showed 5HT2AR-LI-associated mainly with mem-branes of cytoplasmic organelles, including SER and mito-chondria (Figs. 5A,B). Dendritic NMDAR1-LI showed acomparable but largely nonoverlapping cytoplasmic distri-bution to that of 5HT2AR (Fig. 5). In addition, bothreceptors were sometimes distributed along nonsynaptic(Fig. 5C,D) and synaptic portions of plasma membranes indendrites that were contacted by unlabeled axon terminals

(Figs. 5B,D, 6). The gold-silver particles most clearlyidentified 5HT2AR at parasynaptic sites near asymmetricjunctions (Fig. 6B).

Dendrites containing 5HT2AR and/or NMDAR1 receivedsymmetric and asymmetric synapses mainly from axonterminals without detectable immunoreactivity (Figs. 5,6B). The synaptic contacts on these dendrites comprised47.4% (n 5 310) of the total input to immunoreactiveprofiles in the dorsolateral CPN and 66.7% (n 5 309) ofthat seen in the NAc shell. The NMDAR1-IR dendritesreceived more asymmetric synapses from unlabeled axonterminals in the shell of the NAc than in the dorsolateralCPN [0.35 6 0.08/100 µm2 vs. 0.79 6 0.13/100 µm2;F(1,1198) 5 7.344, P 5 0.0068].

Spinous 5HT2AR distributionand relation with NMDAR1

Dendritic spines rarely showed 5HT2AR labeling with orwithout NMDAR1 immunoreactivity and were seen inalmost equal numbers in the dorsolateral CPN and NAcshell (Fig. 4A,C). In spines, 5HT2AR immunoreactivity

Nu

MVB

UD

5HT2A T

M

M

M

SSVsPM

SER

SER

G

Fig. 3. Electron micrograph from the dorsolateral caudate-putamen nucleus (CPN) showing nuclear and cytoplasmic gold label-ing for N-methyl-D-aspartate subtype 1 of glutamate receptor(NMDAR1) within a soma that also contains receptor and immunoper-oxidase reaction product for 5-hydroxytryptamine2A (5HT2A) receptor.The nuclear (Nu) membrane is round and unindented. Immunogold-silver labeling for NMDAR1 receptor within the soma is present alongthe inner nuclear leaflet. In the cytoplasm, the aggregates of immu-noperoxidase reaction product (open arrowheads) appear near or areassociated with saccules of the smooth endoplasmic reticulum (SER)and near the Golgi lamellae (G). Immunogold-silver particles (arrows)are also distributed throughout the cytoplasm and occasionally are

associated with the SER, Golgi, and mitochondrial membranes (M).Boxed area shows a symmetric contact (arrowhead) between a 5HT2AR-labeled terminal (5HT2A T) and the dual-labeled soma. This terminalis also apposed to an unlabeled dendrite (UD). The peroxidase productfor 5HT2AR is seen on the plasma membrane (PM), one small synapticvesicles (SSV), and mitochondrial membranes (M) within the axonterminal. The immunogold particles for NMDAR1 are seen on theinner leaflet of the nucleus (arrows) and in the cytoplasm near the SERand mitochondrial membrane (M). The nuclear membrane is sepa-rated from the plasma membrane by a distance of less than 1 µm.Scale bars 5 1 µm, 0.4 µm in inset.

224 J.J. RODRIGUEZ ET AL.

appeared largely cytoplasmic, but synaptic and nonsynap-tic portions of the plasma membrane also sometimesshowed detectable labeling (Fig. 7). The synaptic inputs tospines containing 5HT2AR immunoreactivity comprisedfewer than 5% of the synaptic inputs to labeled profiles.

In contrast with 5HT2AR, NMDAR1-LI was detectedin many dendritic spines that were seen more often in

the dorsolateral CPN than in the NAc shell (Fig. 4A,B).The NMDAR1 labeling in dendritic spines was mostprominently associated with the spine apparatus andasymmetric postsynaptic junctions but was also seen alongnonsynaptic portions of the plasma membrane. NMDAR1-immunolabeled spines in the dorsolateral CPN received ahigher percentage (44.2%, 137/310) of the total synapticinput to labeled profiles than did the NAc shell (26.9%,83/309). Similarly, more asymmetric synapses were lo-cated on NMDAR1-IR dendritic spines in the dorsolateralCPN than in the shell of the NAc [2.13 6 0.21/100 µm vs.1.32 6 0.16/100 µm2; F(1,1198) 5 9.338, P 5 0.0023].

Axonal 5HT2AR distributionand relation with NMDAR1

5HT2AR-LI was present in selective axon initial seg-ments, small axons, and axon terminals (Figs. 2, 3, 8). Theimmunoreactive initial segments of axons were seen incontinuity with 5HT2AR-labeled somata in favorable planesof section (Fig. 2). These were recognized by their subsur-face coating of electron-dense material, organized arrange-ment of microtubules, and scarcity of rough endoplasmicreticulum (Palay et al., 1968). In the axon initial segments,the peroxidase reaction product showing 5HT2AR-LI waseither diffusely distributed or associated with microtu-bules and/or SER (Fig. 2). In sections processed for duallabeling, immunogold NMDAR1-LI was also seen in axoninitial segments where the receptor was localized to manyof the same organelles (Fig. 2).

The peroxidase and gold markers for 5HT2AR weredistributed along membranes of SSVs and tubulovesiclesand to portions of the plasma membrane in axons and axonterminals (Figs. 2, 3, 8). Immunoreactivity was also seenalong outer mitochondrial membranes when these organ-elles were located near the plasma membranes in largeraxon terminals (Figs. 3A, 8A). There were no regionaldifferences in prevalence and/or subcellular distribution of5HT2AR-LI in axons or axon terminals in the dorsolateralCPN and the NAc shell (Fig. 4A). In sections that wereprocessed for dual labeling, no small axons and few axonterminals contained both 5HT2AR and NMDAR1. NM-DAR1 receptors were present, however, in many smallaxons and terminals and had a subcellular distributionsimilar to that observed in 5HT2AR-containing axons. TheNMDAR1-labeled small unmyelinated axons showed ahigher mean area density in the dorsolateral CPN than inthe shell of the NAc (Fig. 4B).

Axon terminals containing 5HT2AR usually formed syn-apses with dendritic spines and dendrites but also occasion-ally contacted a somata (Figs. 3, 5C). Of all synapticcontacts that were established by 5HT2AR- and/orNMDAR1-labeled axon terminals, 26.3% (10/38) in thedorsolateral CPN and the 35.4% (17/48) in the shell of theNAc were formed by 5HT2AR-labeled axon terminals. Fiftypercent (5/10) of these in the dorsolateral CPN and 35.3%(6/17) in the shell of the NAc were asymmetric synapseswith unlabeled spines. Terminals containing 5HT2AR alsooccasionally formed perforated or nonperforated asymmet-ric synapses with NMDAR1-labeled dendritic spines.

Dendrites that received input from 5HT2AR-containingterminals usually were unlabeled but sometimes alsocontained 5HT2AR or 5HT2AR and NMDAR1 immunoreac-tivities (Fig. 5C). In the NAc shell, 23.5% (4/17) of the5HT2AR-labeled terminals formed symmetric synapses ondually labeled dendrites, whereas none was observed in

Fig. 4. Bar graphs showing the mean area density of 5-hydroxytryp-tamine receptor (5HT2AR)- and N-methyl-D-aspartate subtype 1 ofglutamate receptor (NMDAR1)-labeled neuronal processes in thedorsolateral caudate-putamen nucleus (CPN) and the nucleus accum-bens (NAc) shell. The mean density profiles per unit area is shown forneuronal processes that contain immunoreactivity for (A) 5HT2AR-immunoreactive profiles, (B) NMDAR1-immunoreactive profiles, and(C) dual-labeled profiles. Data were collected from 12 Vibratomesections from each of three animals for a total analyzed surface of5,694.6 mm2 in the dorsolateral CPN and NAc shell. Asterisks indicatea significant difference (P , 0.05).

STRIATAL 5HT2A AND NMDAR1 RECEPTORS 225

B D

CA

5HT2A D

5HT2A G

UT1

UT3

UT2

UT1

UT2

5HT2A T

UT1

UT2

USp

SER

SER

SER

Fig. 5. Dendrites containing immunogold labeling for 5-hydroxy-tryptamine2A receptor (5HT2AR) and immunoperoxidase localization ofN-methyl-D-aspartate subtype 1 of glutamate receptor (NMDAR1) inthe dorsolateral caudate-putamen nucleus (CPN; A,B) and reversedmarkers in the dorsolateral CPN and nucleus accumbens (NAc) shell(C,D). The NMDAR1 labeling is diffusely distributed within thecytoplasm and appears to be most prevalent along plasma membraneand membranes of the smooth endoplasmic reticulum (SER). The5HT2AR labeling is also seen throughout the cytoplasm and alongdiscrete portions of the plasma membrane (arrows, gold particles;open arrowhead, peroxidase). A: A large dual-labeled dendrite receivesa symmetric synapse (arrowhead) from an unlabeled terminal (UT1)and apposes a second terminal (UT2) that forms an asymmetricsynapse (curved arrow) with an unlabeled spine (USp). B: A large

dual-labeled dendrite is apposed to an unlabeled terminal (UT1) andreceives a symmetric synapse (arrowhead) from an unlabeled terminal(UT2). C: A dual-labeled medium–small-diameter dendrite receives asymmetric input (arrowhead) from a 5HT2A-labeled terminal thatshows the immunoperoxidase reaction product associated with theplasma membrane and the membranes of vesicles located at a distancefrom the synapse (open arrow). D: A dual-labeled medium–small-diameter dendrite receives two asymmetric synapses (curved arrows)from unlabeled terminals (UT1–2) and is apposed to a third terminal(UT3). 5HT2AR peroxidase labeling (open arrowhead) is seen beneathUT1 but not UT2. A 5HT2A-labeled glial profile and a dendrite(5HT2A G; 5HT2A D) are located within the neuropil. Scale bars 5 0.4µm in A–C, 0.3 µm in D.

226 J.J. RODRIGUEZ ET AL.

the dorsolateral CPN. No regional differences were ob-served in other types of synaptic contacts. Of the totalsynaptic contacts that were formed by 5HT2AR- and/orNMDAR1-labeled terminals, 73.7% (28/38) in the dorsolat-eral CPN and 64.6% (31/48) in the NAc shell were formedby NMDAR1-immunoreactive terminals. These were

mainly asymmetric axospinous synapses with or withoutrecognizable perforations: 89.3% (25/28) in the dorsolat-eral CPN and 74.2% (23/31) in the NAc shell. The remain-der of the synaptic contacts were formed with dendritesand were mainly asymmetric. The postsynaptic dendriticspines usually were unlabeled and showed no regionaldifferences in distribution.

DISCUSSION

We have shown that in the dorsolateral CPN and in theshell of the NAc, 5HT2A-LI is often localized to cytoplasmiccompartments in somata and medium-sized dendrites butis also present in a few large and small dendrites and indendritic spines, axons, and axon terminals. Althoughmany of the 5HT2AR-immunoreactive somatodendritic pro-files also contained NMDAR1 labeling, the latter receptorwas more often seen in smaller dendrites and dendriticspine. These results suggest coexpression but differentialtargeting of 5HT2A and NMDA receptors in selectivestriatal neurons. Furthermore, we have shown that den-drites containing 5HT2A or 5HT2A and NMDAR1 receptorshave a higher area density but receive a smaller propor-tion of the total synaptic input in the dorsolateral CPNthan in the NAc shell. This observation suggests a moremajor role for 5HT2A receptors in motor functions and/orregional differences in cytoarchitecture or neurotransmit-ter availability.

Methodological considerations

We have referred to the receptor labeling as 5HT2AR-LIand NMDAR1-LI because the 5HT2AR and NMDAR1antisera could recognize other unknown peptide se-quences. We believe, however, that the observed immuno-reactivity accurately represents the distribution of the5HT2A and NMDAR1 receptors. Both antisera that wereused in the present study have been extensively character-ized and shown to be specific by adsorption controls(Petralia et al., 1994; Wu et al., 1998). Selectivity of the5HT2AR and NMDAR1 antisera has been shown by theabsence of cross reaction with other serotonin and gluta-mate receptors, respectively (Petralia et al., 1994; Wu etal., 1998). Furthermore, the regional brain distributions of5HT2AR and NMDAR1 immunoreactivities are similar totheir corresponding mRNA and ligand binding sites (Petra-lia et al., 1994; Pompeiano et al., 1994; Standaert et al.,1994; Wu et al., 1998).

Preembedding immunocytochemistry minimizes the lossof antigens that are sensitive to the process of plasticembedding. This procedure could, however, limit the detec-tion of antigens at a distance from the surface of the tissuedue to incomplete penetration of the immunoreagents(Leranth and Pickel, 1989). Thus, to obtain meaningfulquantitative data, the sections were freeze-thawed toenhance penetration, and ultrathin sections were collectedonly near the resin–tissue interface, having most completeaccess to immunoreagents. Each of the methods used forlocalization of 5HT2A and NMDAR1 receptors has certainadvantages and limitations. Immunogold is less sensitivethan peroxidase but allows more selective subcellularlocalization because of the diffusion of the peroxidasereaction product (Leranth and Pickel, 1989). These differ-ences could contribute to an underestimation of the rela-tive abundance of immunoreactive profiles and/or extent ofcolocalization.

Fig. 6. 5-Hydroxytryptamine2A receptor (5HT2AR) labeling at post-synaptic sites in dendrites (5HT2A D) in the dorsolateral caudate-putamen nucleus (CPN). A: Immunoperoxidase labeling is aggregatednear portions of the plasma membrane (open arrowhead), apposed toan unlabeled terminal (UT). B: Immunogold labeling within and nearan asymmetric synapse (curved arrow) that is formed by an unlabeledterminal (UT). Scale bars 5 0.4 µm.

STRIATAL 5HT2A AND NMDAR1 RECEPTORS 227

Somatodendritic targetingof 5HT2A and NMDAR1 receptors

The present ultrastructural localization of 5HT2AR im-munoreactivity mainly to cytoplasmic organelles in so-mata and medium-sized dendrites of striatal neuronsconfirms and extends results of previous studies showingenrichment of the receptor protein in cytoplasmic fractionsof brain tissue (Laubeya et al., 1986). The prominent5HT2AR labeling of Golgi networks, SER, and other vesicu-lar organelles that are known to play a role in intracellularsynthesis and/or trafficking of receptors (Boudin et al.,

1998; Trimmer, 1999) suggests that most 5HT2AR arelargely nonaccessible to extracellular serotonin and mayundergo activity-dependent mobilization to the plasmamembrane, as has been shown for certain opioid receptors(Shuster et al., 1999). Furthermore, 5HT2AR agonists(Berry et al., 1996) and antagonists (Willins et. al., 1999)increase cytoplasmic 5HT2AR immunoreactivity and pro-duce a redistribution of labeling from apical to largeproximal dendrites. Mobilization in response to high extra-cellular levels of serotonin could contribute to the observedcytoplasmic distribution and infrequent detection of5HT2AR at synaptic or nonsynaptic sites on plasma mem-branes or in smaller dendrites and dendritic spines.

In the present and numerous previous studies, NMDAR1was often seen on synaptic and extrasynaptic plasmamembrane, particularly in smaller dendrites and spines(Aoki et al., 1994; Conti et al., 1997; Gracy and Pickel,1995,1997; Huntley et al., 1994; Petralia et al., 1994, 1997;Wang et al., 1999). The distribution is consistent with theknown enrichment of NMDAR activation-responsive phos-phoproteins (NARPPs) in postsynaptic densities (Scheetzand Constantine-Paton, 1996; Schen et al., 1998). We alsooccassionally saw 5HT2AR labeling within or near asymmet-ric striatal synapses, and this receptor has been localizedto postsynaptic densities in the rat cerebral cortex andolfactory system (Hamada et al., 1998). Thus, the postsyn-aptic densities are likely sites where activation of 5HT2ARcan potentiate NMDA-mediated opening of ion channels(Blank et al., 1996; Kornau et al., 1995).

Interestingly, we also saw NMDAR1 immunogold label-ing discretely localized to plasma membranes of selectiveneurons that showed cytoplasmic NMDAR1 immunoreac-tivity. During development, NARPPs are associated withneuronal nuclei (Scheetz and Constantine-Paton, 1996),suggesting that nuclei may also be functional bindingsites. The role, if any, of NMDAR1 receptors on nuclearmembranes is unknown but could reflect sites that areinvolved in NMDA-mediated calcium-induced gene tran-scription and/or apoptotic cell death (Bading et al., 1997;Cheung et al., 1998; Hardingham et al., 1998).

Axonal localizationof 5HT2A and NMDA receptors

We observed 5HT2AR and/or NMDA immunoreactivitywithin a few axon initial segments and in small axons andaxon terminals. The rarity of 5HT2AR in these profiles mayreflect limited detectabity of the antigen in axons or

Fig. 7. N-methyl-D-aspartate subtype 1 of glutamate receptor(NMDAR1) and/or 5-hydroxytryptamine2A receptor (5HT2AR)-labeleddendritic spines within the nucleus accumbens (NAc) shell. A: Immu-noperoxidase labeling in a dendritic spine (5HT2A Sp) is seen withinthe thick postsynaptic density located beneath an unlabeled terminal(UT1) and along the extrasynaptic plasma membrane (open arrow-heads). The spine appears to have arisen from a 5HT2A-labeleddendrite (5HT2A D; open arrowhead). The dendrite receives a symmet-ric synapse (arrowhead) from an unlabeled terminal (UT2). Anunlabeled spine (USp) is seen in the neuropil and receives anasymmetric synapse (curved arrow) from an unlabeled terminal(UT3). B: Immunoperoxidase labeling for 5HT2AR (open arrowhead)within the postsynaptic density beneath an unlabeled terminal (UT1)in a dendritic spine that also contains gold particles for NMDAR1(arrows) within the neck region. NMDAR1 immunogold is also seenalong the plasma membrane of a nearby dendrite (NMDAR1 D) thatreceives a synapse (curved arrow) from another unlabeled terminal(UT2). Scale bars 5 0.4 µm.

228 J.J. RODRIGUEZ ET AL.

transport of 5HT2AR to extrinsic terminal fields. In addi-tion, within the CPN and NAc shell, approximately half ofthe terminals containing 5HT2AR immunoreactivity formedasymmetric synapses that are typical of glutamatergic

axons (Gundersen et al., 1996). In the cerebral cortex,5HT2AR have been localized immunocytochemically topresynaptic axons and dendrites (Jakab and Goldman-Rakic, 1998). Furthermore, 5HT2AR are thought to play arole in an atypical mode of presynaptic glutamate release,thereby contributing to asynchronous postsynaptic poten-tial in pyramidal neurons (Aghajanian and Marek, 1999).These results and the present finding of colocalization of5HT2A and NMDA receptors in dendrites of the CPN andNAc shell strongly implicate 5HT2AR in direct and indirectmodulation of the output from spiny striatal neurons,most of which are gaminobutyric acid-ergic (Ribak et al.,1979).

Regional comparison of 5HT2ARand NMDAR1 localization

We have shown by electron microscopy that more den-drites per unit area contain 5HT2AR or 5HT2AR andNMDAR1 immunoreactivities in the dorsolateral CPNthan in the NAc shell. In contrast, equal or higher levels of5HT2A receptor mRNA and immunoreactivity have beenreported by light microscopy in these regions (Morilak etal., 1993, 1994; Pompeiano et al., 1994; Wright et al., 1995;Mijnster et al., 1997). These results are not necessarilydiscrepant because certain spiny projection neurons in theNAc core, which is in many ways equivalent to the CPN,are known to have more spines and dendritic branchpoints than those in the NAc shell (Meredith et al., 1992).Thus, although individual cells may contain equivalentlevels of 5HT2AR mRNA and/or protein in the NAc shellthan in the CPN, more labeled dendrites are likely to becut in a single plane of section. In addition, the smallernumber 5HT2AR-labeled profiles in the NAc shell as com-pared with the CPN may be attributed to downregulationof the receptor in the NAc shell, which has a denserserotonergic innervation and a higher serotonin concentra-tion than the motor core (Deutch et al., 1992; Van Bocks-taele and Pickel, 1993). Potential differences in serotoner-gic input are suggested in the present study by thedemonstration that 5HT2AR-immunoreactive dendrites inthe NAc shell received a greater proportion of the totalsynaptic input to labeled dendrites than did those in theCPN. Despite these differences, the greater number ofdendrites containing 5HT2A or 5HT2A and NMDA receptorsin the dorsolateral CPN versus NAc shell suggests a morecritical role for these receptors in motor function (Kulikovet al., 1995; Forrest et al., 1996; Hillegaart et al., 1996;Miyawaki et al., 1997).

ACKNOWLEDGMENT

We thank Dr. Adena L. Svingos for her critical commen-tary of this paper.

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