Pattern of expression of the serotonin2C receptor messenger RNA in the basal ganglia of adult rats

Pattern of Expression of the Serotonin2CReceptor Messenger RNA in the Basal

Ganglia of Adult Rats

K. EBERLE-WANG, Z. MIKELADZE, K. URYU, AND M.-F. CHESSELET*Department of Pharmacology, University of Pennsylvania,

Philadelphia, Pennsylvania 19104

ABSTRACTThe distribution of the serotonin (5-HT) receptor 5-HT2C mRNA was examined at the

single-cell level with in situ hybridization histochemistry and emulsion autoradiography inthe basal ganglia and mesolimbic system of adult rats, with focus on the pallidum and thesubstantia nigra, which receive striatal inputs and play a critical role in basal gangliafunction. 5-HT2C receptor mRNA expression was always restricted to a subpopulation ofneurons in the regions examined. In the neostriatum, labeled neurons were more numerous inthe rostral nucleus accumbens than in the caudal nucleus accumbens and were morenumerous in the ventral and ventrolateral caudate-putamen than in the dorsal caudate-putamen, where labeled neurons were restricted to isolated clusters. In striatal target areas,dense labeling in the entopeduncular nucleus (internal pallidum, direct striatal outputpathway) contrasted with an absence of labeling in the globus pallidus (external pallidum,indirect striatal output pathway). Double-label in situ hybridization in the substantia nigrarevealed coexpression of 5-HT2C receptor mRNA with glutamic acid decarboxylase but notwith tyrosine hydroxylase mRNA, indicating that it was restricted to g-aminobutyric acid(GABA)ergic neurons. In this region, dense labeling for 5-HT2C mRNAwas found in half of theneurons at middle and caudal levels of both the pars compacta and the pars reticulata, withlittle labeling rostrally. The data suggest that drugs acting on the 5-HT2C receptor couldselectively affect discrete neuronal populations in the basal ganglia and mesolimbic systemsand indicate a new level of neurochemical heterogeneity among GABAergic neurons of thesubstantia nigra. J. Comp. Neurol. 384:233–247, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: serotonin; in situ hybridization; digoxigenin; nucleus accumbens; substantia nigra

The neurotransmitter serotonin (5-hydroxytryptamine;5-HT) exerts widespread effects in the mammalian centralnervous system and is involved in the regulation of numer-ous functions, including motor behavior (Palacios et al.,1991; Jacobs and Fornal, 1993; Frazer and Hensler, 1994).The effects of 5-HT are mediated by a variety of receptorsubtypes, which are characterized by different pharmaco-logical properties and are encoded by different genes(Harrington et al., 1992; Frazer andHensler, 1994; SaudouandHen, 1994).Aprerequisite to understanding the role ofserotonergic systems in the brain is the determination ofthe pattern of expression of these receptor subtypes inregions that receive serotonergic innervation.The basal ganglia, a group of subcortical brain regions

rich in serotonergic innervation in both rodents (Stein-bush, 1981) and primates (Lavoie and Parent, 1990), areknown to play a role in the regulation of motor behavior.Although the mRNAs encoding several types of serotoner-gic receptors have been detected in the basal ganglia

(Saudou and Hen, 1994), the mRNA encoding the 5-HT2Creceptor, a serotonergic receptor subtype originally de-scribed in the choroid plexus (Pazos et al., 1984; Conn etal., 1986), is of particular interest, because marked differ-ences have been noted in the distribution of this mRNAamong subregions of the basal ganglia (Hoffman andMezey, 1989; Mengod et al., 1990; Wright et al., 1995).Furthermore, behavioral studies in rats have suggestedthat the 5-HT2C receptor is involved in the control of motorbehavior (Lucki et al., 1989; Curzon and Kennett, 1990;Eberle-Wang et al., 1996a,b).

Grant sponsor: Public Health Service; Grant numbers: MH-48125,MH-44894; Grant sponsor: Tourette SyndromeAssociation.*Correspondence to: Marie-Francoise Chesselet, M.D., Ph.D., Depart-

ment of Neurology, UCLA School of Medicine, 710 Westwood Plaza, LosAngeles, CA90095. E-mail: [email protected] 2April 1996; Revised 21 February 1997;Accepted 7March 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 384:233–247 (1997)

r 1997 WILEY-LISS, INC.

Heterogeneities in the distribution of the 5-TH2C recep-tor mRNA suggest that drugs acting on this receptor mayselectively affect some neuronal populations in the basalganglia. However, the details of this heterogeneous distri-bution remain unknown, hampering the interpretation offunctional studies of the effects of drugs acting on 5-HT2Creceptors. In particular, little is known about the detaileddistribution of this receptor mRNA in regions of the basalganglia that receive inputs from the striatum, such as thepallidum and the substantia nigra. This information wouldbe of particular interest, because output neurons of theseregions play a critical role in regulating motor behavior(Albin et al., 1989; Chesselet and Delfs, 1996).The goal of the present study was to further characterize

the distribution of the 5-HT2C receptor mRNA in the basalganglia at the single-cell level, with particular emphasison the pallidum and the substantia nigra. Data on thenucleus accumbens and the ventral tegmental area areincluded for comparisonwith the striatumand the substan-tia nigra pars compacta, respectively. Brain sections ofadult rats were processed for in situ hybridization histo-chemistry with a 35S-radiolabeled probe complementary tothe third intracytoplasmic loop of the 5-HT2C receptormRNA, and the hybrids were detected at the single-celllevel by emulsion autoradiography. In the ventral mesen-cephalon, double in situ hybridization experiments wereperformed to determine whether 5-HT2C receptor mRNAwas expressed by dopaminergic or g-aminobutyric acid(GABA)ergic neurons. In addition, the pattern of expres-sion of the mRNA in the substantia nigra was comparedwith the density of serotonergic nerve terminals deter-mined by radioligand autoradiography.

MATERIALS AND METHODS

Tissue preparation

Adult male Sprague-Dawley rats (250–300 g) werehoused under standard conditions and were killed bydecapitation at approximately 10 a.m. Brains were quicklyfrozen on powdered dry ice and were cut with a cryostat insections (10 µm), which were thaw-mounted onto gelatin-coated slides and stored at 270°C. The experimentalprotocols were approved by the institutional Animal CareCommittee and conformed to NIH Standards on the Careand Use of Animals in Research.

In situ hybridization histochemistry

Radiolabeled antisense and sense cRNAs were tran-scribed from cDNAs, as previously described (Chesselet etal., 1987), by using appropriate polymerases, 2.5 µM35S-UTP (1,000 Ci/mmol; New England Nuclear, Boston,MA) dissolved in 10 µM cold S-UTP, with ATP, CTP, andGTP in excess. The template cDNA for the 5-HT2C receptorconsisted of a 202 base-pair sequence isolated by polymer-ase chain reaction with oligonucleotide primers based onthe published sequence of the 5-HT2C receptor mRNA (agift from D. Pritchett, University of Pennsylvania). ThecDNAcorresponded to nucleotides 881–1,083 of the 5-HT2Creceptor cDNA, a sequence that codes for the presumedthird intracytoplasmic loop of the 5-HT2C receptor (Juliuset al., 1988). A current sequence comparison of the 5-HT2Creceptor cDNA probe with the NCBI BLAST program(Altschul et al., 1990) (using the default parameters)confirmed the 100% sequence identity of this probe withthe rat 5-HT2C receptor mRNA, with no reported sequence

identities with any other 5-HT receptor subtypes in thenonredundant database. Both sense and antisense RNAprobes were synthesized. The full length of the probe wasconfirmed by denaturing gel electrophoresis.To identify the location of dopamine-containing neurons

of the substantia nigra pars compacta, serially adjacentsections through the ventral mesencephalon were pro-cessed for in situ hybridization with the 5-HT2C receptorcRNAprobe and a probe for tyrosine hydroxylase (TH), thelimiting enzyme of catecholamine synthesis. The cDNAused to synthesize the TH probe was a 110 base-pairfragment of the cDNA clone pTHu, which was isolatedfrom PC12 cells (Lewis et al., 1983). This template, whichwas generously provided by Drs. E. Lewis and D. Chikarai-shi, corresponds to the Kpnl (base 1520)-Bal I (base 1,407)fragment of rat TH cDNA (Grima et al., 1985).In situ hybridization was carried out as previously

described (Chesselet et al., 1987; Chesselet, 1990). Briefly,tissue sections were brought to room temperature anddried rapidly under a stream of cold air, postfixed in 3%paraformaldehyde containing 0.02% diethylpyrocarbonate(DEPC), acetylated, and dehydrated. Sections were incu-bated with 8–10 ng of probe (approximately 400,000dpm/ng) in humid chambers at 50°C for 3.5 hours. Afterhybridization, nonspecific hybridization was reduced bywashing in 50% formamide in 2 3 standard sodium citrate(2 3 SSC; 0.3 M NaCl/0.03 M sodium citrate) at 52°C andtreatment with RNAse A (100 µg/ml) at 37°C for 30 3minutes. After overnight incubation in 2 3 SSC/0.05%Triton X-100, slides were dehydrated in graded ethanol,defatted in xylene, and coated with Kodak NTB3 emulsiondiluted 1:1 with 300 mM ammonium acetate. Sectionswere exposed for 6–8 weeks and were developed in KodakD19 developer at full strength, lightly counterstained withhematoxylin and eosin, and mounted with Eukitt (Cali-brated Instruments, Hawthorne, NY).To examine the neurochemical identity of cells in the

ventral mesencephalon that express 5-HT2C receptormRNA, double-labeling experiments were performed onsections serially adjacent to those processed for the single5-HT2C receptor detection. These sections were hybridizedsimultaneously with both a 35S-radiolabeled 5-HT2C recep-tor mRNA probe and a digoxigenin-labeled RNA probe foreither TH or glutamic acid decarboxylase (Mr 67,000:GAD67), a key enzyme in GABA synthesis (Erlander andTobin, 1991). Synthesis of the TH andGAD67 cRNAprobeswas achieved, as previously described (Mercugliano et al.,1992), with digoxigenin-labeled UTP (Boeringher-Mann-heim, Indianapolis, IN). After the overnight wash in 2 3SSC and Triton X-100, the sections were processed fordetection of the digoxigenin-labeled hybrids with an antidi-goxigenin antibody coupled to alkaline phosphatase (Lewisand Baldino, 1990), as described previously (Chesselet,1990), except that the enzymatic reaction was stopped in100 mM Tris HCl, pH 9.5, 100 mM NaCl, and 50 mMMgCl2, followed by dehydration, quick clearing in xylene,and coating with Ilford nuclear research emulsion K.5D(Polysciences,Warrington, PA).After exposure (6–8weeks),the sections were developed as described above, mountedwith an aqueous medium (Biomeda, Foster City, CA), andimmediately analyzed.

Specificity of hybridization and controls

The 5-HT2C receptor cDNA that was used as a templatefor probe synthesis has been shown to hybridize specifi-

234 K. EBERLE-WANG ET AL.

cally to a single mRNA band on Northern blots (D.Pritchett, personal communication). Tissue sections hybrid-ized with sense probes or without the antidigoxigeninantibody were processed in parallel with those hybridizedto the antisense probes to control for the specificity ofhybridization and detection. In addition, the specificity ofthe anatomical distribution of the labeling was carefullyassessed in each experiment (see Results).

Cyanoimipramine binding

Quantitative autoradiography of 3H-cyanoimipraminebinding sites, a marker of serotonergic uptake sites (Wolfeet al., 1987), was performed according to the method ofKovachich et al. (1988). Briefly, sections were desiccated at220°C, brought to room temperature under a stream ofcold air, and incubated in 30 ml of Tris/saline buffer (50mM Tris, 150 mM NaCl, pH 7.4) containing 0.5 nM3H-cyanoimipramine (American Radiolabelled Chemicals,St. Louis, MO; 83.6 Ci/mmol, 1mCi/ml) at 4°C for 24 hours.Nonspecific binding was defined in the presence of 100 µMdesmethylimipramine (DMI; Sigma, St. Louis, MO). Afterthe incubation, the sections were transferred to a freshbuffer and were washed for 60 minutes at 4°C. Following aquick rinse in ice-cold distilled water, the sections weredried on a slide warmer and exposed with autoradio-graphic standards to [3H]Ultrofilm for 3 weeks at roomtemperature. The films were developed as described above.

Data analysis

Sections processed for in situ hybridization histochemis-try were examined under darkfield and brightfield illumi-nation with a Leitz Aristoplan microscope. Only sectionsprocessed in parallel in the same experiment were used forquantitative comparison. Furthermore, in all experi-ments, only one section was apposed to each glass slide tominimize possible variability in the autoradiographic sig-nal generated with emulsion autoradiography (Chesseletand Weiss-Wunder, 1994). This limits the number ofsections per animal that can be compared quantitativelybut, according to our experience, results in very reliabledata. The charting of labeled neurons was accomplishedunder darkfield illumination with an Olympus microscopeequipped with a drawing tube.The density of labeled neurons was measured at two

levels of the substantia nigra (rostral, 4.20 mm frominteraural line; caudal, 3.40 mm from interaural line;Paxinos and Watson, 1986) in eight rats. Labeled neuronspresent in the substantia nigra pars compacta and parsreticulata in both hemispheres were counted on cameralucida drawings of one section per rat at each level. Thesurface area of the structure examined was measured witha MORPHON image-analysis system (Philadelphia, PA)(Mercugliano et al., 1992), and data were averaged foreach level. In addition, the proportion of neurons express-ing 5-HT2C receptor mRNA at the same two levels of thesubstantia nigra was obtained by counting labeled andunlabeled neurons within five microscope fields over thepars compacta and the pars reticulata of the substantianigra in two sections from five additional rats that wereprocessed in a separate experiment. In both cases, neuronswere identified on the basis of the presence of a visiblenucleus, and labeled cells were defined as cells coveredwith a cluster of reduced silver grains, which were clearlydistinguishable from background. Stereological methods,as discussed previously (Saper, 1996), are not necessary

for the purpose of the data analysis in this study, whichonly provides an estimate of the relative number of labeledneurons in the conditions of the experiment.Analysis of film autoradiograms for 3H-cyanoimipra-

mine binding sites was performed by using the Dumasimage-analysis system (Drexel University, Philadelphia,PA) with the BRAIN software package (NIH, Bethesda,MD). Plastic-embedded tritium standards, calibratedagainst 3H-containing brain-mash sections, were used forquantification. Specific binding was determined by sub-tracting the value for nonspecific binding from total bind-ing for each region of interest. Labeling density wascompared with an unpaired, two-tailed Student’s t-test.The threshold for significance was P , 0.05.

RESULTS

Sections incubated with the antisense 35S-radiolabelledprobe for the 5-HT2C receptor exhibited a strong autoradio-graphic labeling over the choroid plexus (Fig. 1D) andnumerous other brain areas, including the hippocampus,the nucleus accumbens, the striatum (Fig. 1A,C), thesubthalamic nucleus (Fig. 5B), the claustrum (Fig. 2A–C),and the lateral septum (Fig. 2D–F). Sections hybridizedwith the 35S-radiolabeled sense probe did not show anyspecific labeling (Fig. 1B). In the basal ganglia, labeledareas included the ventral part of the caudate nucleus, theentopeduncular nucleus, the subthalamic nucleus, and thesubstantia nigra.

Pattern of expression of 5-HT2C receptormRNA in the striatum and nucleus

accumbens

Clusters of silver grains were found over individualneurons in the striatum (Fig. 1A) and the nucleus accum-bens (Fig. 1C) and were in sharp contrast to the diffuse,low autoradiographic background seen in sections hybrid-ized with sense (control) RNA probes (Fig. 1B). Labeledneurons were found in discrete areas of the caudate-putamen, mostly in the ventral and ventrolateral parts ofthe nucleus (Figs. 1A, 2). Occasional groups of labeledneurons were seen in the medial striatum and along itsventricular border. In the dorsolateral striatum, specificlabeling was found in clusters of cells more frequent in thecaudal aspect of this region at the level of the anteriorcommissure (Fig. 2F). Labeled neurons were also presentthroughout the whole rostrocaudal extent of the nucleusaccumbens (Fig. 2), both in the shell and in the core (Fig.2). Labeled neurons were more numerous in the rostralpart of the nucleus (Fig. 2A) than at more caudal levels(Fig. 2B–E). In both the striatum and the nucleus accum-bens, most labeled neurons were medium-sized round oroval cells with a very thin band of lightly stained cyto-plasm surrounding a smooth, round nucleus (Fig. 1C,arrow), a morphology that is typical of efferent neurons(Parent and Hazrati, 1995). Specific labeling was notdetected in large neurons or in glial cells.

Pallidum and subthalamic nucleus

Expression of the 5-HT2C receptor mRNA was dense incells of the entopeduncular nucleus (Fig. 3A,B), which isthe equivalent of the internal pallidum in primates. Incontrast, labeled cells were conspicuously absent from theglobus pallidus (Fig. 3C), which is the equivalent of theexternal pallidum in primates, revealing a major differ-

5-HT RECEPTOR mRNA IN BASAL GANGLIA 235

Fig. 1. A: Low-power, darkfield photomicrograph of labeling for theserotonin (5-HT)2C receptormRNAin a coronal section of the ventrome-dial striatum that was taken 8.7 mm rostral to interaural zero.Clusters of silver grains over labeled cells (arrows) were in contrast tothe light, nonspecific background labeling that was distributed uni-formly throughout sections processed with the sense (control) probeshown in B. C: High-power, brightfield photomicrograph of cells

expressing 5-HT2C receptor mRNA in the nucleus accumbens (arrow)and unlabeled glial cells (arrowhead). Note that the lightly counter-stained cytoplasm cannot be distinguished on the black-and-whitephotomicrograph.D:Low-power, darkfield photomicrograph of denselylabeled cells in the choroid plexus. chp, choroid plexus. Scale bars 5200 µm inA,B, 10 µm in C, 50 µm in D.


Fig. 2. Schematic diagram of the distribution of neurons express-ing the 5-HT2C receptor in the forebrain of adult rats. Hemicoronalsections are shown from rostral to caudal (A–F). Dots represent thelocations of labeled cells, as observed in sections from several animals,without attempts to correlate the density of the dots to the number oflabeled cells. Weakly labeled neurons in the cerebral cortex are notillustrated. Acb, accumbens; AcbC, accumbens core; AcbSh, accum-

bens shell; AOP, anterior olfactory nucleus, posterior; Cg3, cingulatecortex, area 3; CPu, caudate putamen; Cl, claustrum; DP, dorsalpeduncular cortex; FStr, fundus striati; IL, infralimbic cortex; SD,lateral septal nucleus, dorsal; LSI, lateral septal nucleus, intermedi-ate; LSV, lateral septal nucleus, ventral; Pir, piriform cortex; TT,taenia tecta; VP, ventral pallidum. Numbers indicate distance in mmfrom interaural zero. Scale bar 5 1 mm.

ence in the expression of the 5-HT2C receptor mRNAbetween the two pallidal segments.The subthalamic nucleus, as previously reported (Hoff-

man and Mezey, 1989; Mengod et al., 1990; Wright et al.,1995), is a region that plays a major role in the generationof hyperkinetic movements. It contained large numbers ofcells that expressed a high level of mRNA for 5-HT2Creceptors (Fig. 5B).

Ventral mesencephalon

The greatest numbers of labeled neurons in the ventralmesencephalon were found in the substantia nigra. How-ever, their density varied dramatically at different rostro-caudal levels of the nucleus. The distribution of neuronsexpressing 5-HT2C receptor mRNA is illustrated in frontaland sagittal sections of the substantia nigra in Figures 4and 5, respectively. In both Figure 4 and Figure 5, thestippled areas indicate the location of the substantia nigrapars compacta, as identified in adjacent sections by thepresence of a high density of neurons expressing THmRNA. Neurons labeled for the 5-HT2C receptor mRNAwere present in both the substantia nigra pars compactaand pars reticulata at all rostrocaudal and mediolaterallevels examined (Figs. 4–6). Their density, however, wasmarkedly higher caudally than rostrally, in both thesubstantia nigra pars compacta and pars reticulata (Figs.4–6). In the rostral substantia nigra (4.20 mm frominteraural zero; Paxinos and Watson, 1986), the density oflabeled neurons was 3.16 6 0.7 neurons per mm2 (mean 6S.E.M.; n 5 8), whereas, in a caudal section of the samerats (3.40 mm from interaural zero), the density of labeledneurons was 36.32 6 2.7 neurons per mm2 (mean 6S.E.M.; n 5 8; P 5 0.0001; unpaired two-tailed Student’st-test).Even in the caudal regions of the substantia nigra,

which contain numerous neurons expressing the 5-HT2CmRNA, many neurons remained unlabeled even after longautoradiographic exposure (Fig. 6). To confirm this visualobservation, the proportions of labeled and unlabelledneurons were measured in the rostral and caudal substan-tia nigra in five additional animals. When both parscompacta and reticulata were considered together, out of atotal of 100.4 6 2.6 neurons, 7.0 6 0.9% were labelled inthe rostral sections vs. 50.14 6 3.0% out of 145.8 6 12.5 inthe caudal sections (mean 6 S.E.M.; n 5 5; P 5 0.0001;unpaired two-tailed Student’s t-test). Similar differencesin labelled cells were found in the pars compacta (8.8 61.4% out of 52.86 2.0 neurons rostrally vs. 47.76 1.5% outof 63.8 6 4.1 neurons caudally; mean 6 S.E.M.; n 5 5; P 50.0001; unpaired two-tailed Student’s t-test) and in thepars reticulata (3.5 6 1.0% out of 62 6 6.5 neuronsrostrally vs. 49.8 6 4.5% out of 107.4 6 7.6 neuronscaudally; mean 6 S.E.M.; n 5 5; P 5 0.0001; unpairedtwo-tailed Student’s t-test).A fewmoderately labeled neurons expressing the 5-HT2C

receptor mRNA were present in all major subdivisions ofthe ventral tegmental area (Fig. 7), with a notably highdensity of labeled cells in the caudal linear nucleus of theraphe (Fig. 7D,E). Caudal to the substantia nigra, labeledneurons were present in the retrorubral field in the regionof theA8 dopaminergic cell group (Fig. 7E).In the rat, only a fraction of neurons located in the

substantia nigra pars compacta and in the ventral tegmen-tal area are dopaminergic. Therefore, double-labeling insitu hybridization experiments were performed to deter-mine whether the neurons that expressed the 5-HT2Creceptor mRNA also contained the mRNA encoding TH,the rate-limiting enzyme of catecholamine synthesis. De-spite the presence of neurons labeled for 5-HT2C receptormRNAnext to THmRNA-positive neurons (Fig. 8), double-labeled cells were never observed in the substantia nigrapars compacta (Fig. 8), the ventral tegmental area, or the

Fig. 3. A: Brightfield photomicrograph of neurons expressing5-HT2C receptor mRNA in the entopeduncular nucleus (EP). B:Darkfield photomicrograph of heavily labeled cells in the EP incontrast to the absence of labeling in the globus pallidus (GP), which isshown at the same magnification inC. Scale bars 5 20 µm.


linear nuclei of the raphe (not shown). Similarly, the fewTH mRNA-positive neurons located ventrally in the sub-stantia nigra pars reticulata did not express the 5-HT2C

receptor mRNA (data not shown). The A8 dopaminergiccell group was not examined in the double-label experi-ments.

Fig. 4. A–D: Schematic diagram of the distribution of neuronsexpressing tyrosine hydroxylase (TH) and 5-HT2C receptor mRNAs infrontal sections of the substantia nigra. The schematic coronal sec-tions to the far right highlight the level of the substantia nigra (box)analyzed in the first three columns. The left column (TH) illustratesthe location of neurons expressing TH mRNA; the middle column(5-HT2C) illustrates the location of neurons expressing the mRNAencoding the 5-HT2C receptor; and the right column (5-HT2C 1 TH)illustrates the location of both mRNAs. In each of the first three

columns, the dots represent neurons expressing the mRNAs, asobserved in sections from several animals, without attempts tocorrelate the density to the number of labeled cells. The stippled areasin the right column indicate the location of the TH-positive cell bodiesof the pars compacta shown on the left column. Note that labeled cellbodies are present in both the pars compacta and the pars reticulata,but they are much more numerous at caudal levels than at rostrallevels. Numbers indicate distance from interaural zero in mm. Scalebar 5 1 mm.


By contrast, double-label experiments for GAD and5-HT2C receptor mRNAs confirmed that 5-HT2C receptormessage was expressed within neurons that also stainedpositively forGADmRNA(Fig. 9, arrowheads).All cellswithinthe substantia nigra that were labeled for 5-HT2C receptormRNA were GAD-positive. However, not all GAD-positivecells expressed the 5-HT2C receptor mRNA (Fig. 9, arrow).To determine whether the marked rostrocaudal differ-

ences in the density of cells expressing the 5-HT2C receptormRNA in the substantia nigra paralleled differences in thedistribution of serotonergic terminals, sections from ros-tral (4.20 mm from interaural line) and caudal (3.40 mmfrom interaural line; Paxinos and Watson, 1986) substan-tia nigra were processed for quantitative autoradiographyof 3H-cyanoimipramine binding, a marker of 5-HT uptake.Specific 3H-cyanoimipramine bindingwas present through-out the substantia nigra, and there was no significantdifference in the density of 3H-cyanoimipramine bindingsites in rostral vs. caudal substantia nigra [rostral 56.4 62.9 mCi/ml (n 5 9) vs. caudal 67.4 6 5.0 mCi/ml (n 5 9);

mean 6 S.E.M.; P 5 0.1463; unpaired two-tailed Student’st-test].

DISCUSSION

The data of the present study reveal that the markedheterogeneities in the distribution of the 5-HT2C receptormRNApreviously observed in the striatum also character-ize other regions of the basal ganglia. Not only was thereceptor mRNAexpressed in some regions (e.g., entopeduncu-lar nucleus) and not others (globus pallidus), but its level ofexpression varied markedly within labeled regions. Notably,the expression of the 5-HT2C receptor mRNA in the ratsubstantia nigra was confined to GABAergic neurons locatedin the caudal part of both pars compacta and pars reticulata.

Heterogeneous distribution of 5-HT2C

receptor mRNA in the striatum and pallidum

The general pattern of labeling observed in this studywas similar to that obtained previously with a variety of

Fig. 5. A–C: Schematic diagram of the distribution of neuronsexpressing TH and 5-HT2C receptor mRNAs in sagittal sections of thesubstantia nigra. The schematic sagittal sections to the far righthighlight the level of the substantia nigra (box) analyzed in the leftand center columns. Dots represent the location of neurons expressingthe 5-HT2C receptor mRNA, as observed in sections from severalanimals, without attempts to correlate the density of the dots to the

number of labeled cells. The stippled areas in the right columnindicate the location of the TH-positive cell bodies of the pars compactashown on the left column. Note that labeled cell bodies are present inboth the pars compacta and the pars reticulata, but they are muchmore numerous at caudal levels than at rostral levels. STh, subtha-lamic nucleus. Numbers indicate distance from the midline in mm.Scale bar 5 0.5 mm.


Fig. 6. A: Low-power, darkfield photomicrograph of neurons la-beled for the 5-HT2C receptor mRNA(arrow) in the rostral aspect of thesubstantia nigra. B: Low-power, darkfield photomicrograph of neu-rons labeled for the 5-HT2C receptor mRNA (arrow) in the caudalaspect of the substantia nigra. C,D: High-power, brightfield photomi-

crographs of densely labeled cells (arrows) in the caudal substantianigra pars compacta (C) and in the substantia nigra pars reticulata(D). Arrowheads indicate unlabeled glia cells, and the star in Didentifies an unlabeled neuron. pc, Pars compacta; pr, pars reticulata.Scale bars 5 100 µm inA,B, 20 µm in C,D.


different probes for the 5-HT2C receptor mRNA (Hoffmanand Mezey, 1989; Mengod et al., 1990; Wright et al., 1995),further supporting the specificity of the hybridizationpattern reported here. In the striatum, most of the specificlabeling was found over scattered groups of neurons.Although previous studies using film autoradiograms havereported a large population of lightly to moderately labeledcells expressing 5-HT2C receptor mRNA uniformly distrib-uted throughout specific levels of the striatum (Numan etal., 1995; Wright et al., 1995), in our studies using emul-sion autoradiography, this light, more diffuse labeling wasnot associated with identified neuronal cell bodies and is

likely to represent background hybridization (see Fig. 1A).It cannot be excluded, however, that a very low level of5-HT2C receptor mRNA is present in striatal neurons thatwere unlabelled in the present study.A positive correlation between 5-HT2C binding sites and

receptor mRNA was particularly clear in the case of thecaudate-putamen, where neuronal cell bodies expressingthe 5-HT2C receptor mRNA were more abundant in theventral and ventrolateral parts of the nucleus, areas thatare rich in 5-HT2C binding sites (Mengod et al., 1990). Thelack of cellular resolution with quantitative receptor auto-radiography does not allow one to determine with cer-

Fig. 7. A–E: Schematic diagram of the distribution of neuronsexpressing THmRNAand 5-HT2C receptormRNAin frontal sections ofthe ventral tegmental area. The schematic coronal sections to the farright highlight the level of the ventral tegmental area (box) analyzedin the left and center columns. Numbers indicate distance frominteraural zero in mm. Dots represent the location of neurons express-ing the 5-HT2C receptor mRNA, as observed in sections from several

animals, without attempts to correlate the density of the dots to thenumber of labeled cells. A8, dopamine cell group in the retrorubralfield; CLi, caudal linear nucleus raphe; IF, interfascicular nucleus;PBP, parabrachial pigmented nucleus; PN, paranigral nucleus; RLi,rostral linear nucleus raphe; VTA, ventral tegmental area. Scale bar(in D) 5 500 µm.


tainty whether the same neurons express the mRNA andthe corresponding receptor. The similar distribution ofmRNA and binding sites, however, strongly supports thehypothesis that 5-HT2CmRNAdetected in these neurons istranslated into receptor protein. The ventral striatum isthe striatal region that receives the highest density ofserotonergic nerve terminals in the rat (Steinbush, 1981).The data suggest that the 5-HT2C receptor may mediatesome of the effects of 5-HT released from afferent neuronsto this region originating in the dorsal raphe nucleus (VanBockstaele et al., 1993).The 5-HT2C receptor mRNAwas present in both the core

and the shell of the nucleus accumbens, where labeledneurons were distributed in a decreasing rostrocaudalgradient. Conspicuous differences in the effect of cholecys-tokinin or dopaminergic receptor stimulation have beendescribed between the rostral and the caudal nucleusaccumbens, supporting the existence of anatomical andfunctional differences between these regions (Vaccarinoand Rankin, 1989; Essman et al., 1993; Voorn et al., 1994).Local infusions of 5-HT into the nucleus accumbens de-crease locomotor activity (Jones et al., 1981); however, it isunknown whether or not regional differences in sensitivityto serotonergic agents occur within the nucleus accum-bens. The organization of inputs and outputs differs mark-edly among subregions of the nucleus accumbens (Ber-endse et al., 1992; Zahm and Heimer, 1993). Thetopographical heterogeneity of the 5-HT2C distributionsuggests that pharmacological agents acting on this recep-

tor subtype may preferentially influence functions medi-ated by the rostral vs. the caudal nucleus accumbens.Labeling for the 5-HT2C receptor was dense in most

neurons of the entopeduncular nucleus (internal pallidum)but was virtually absent from the globus pallidus (externalpallidum). The entopeduncular nucleus and the substan-tia nigra pars reticulata comprise the two major outputnuclei of the basal ganglia (Parent and Hazrati, 1995).They receive inputs from the caudate-putamen directly aswell as indirectly, by way of the globus pallidus (Albin etal., 1989). Therefore, the differential distribution of 5-HT2Creceptor mRNAmay be indicative of differential regulationof the direct and indirect pathways of striatal output by5-HT. This is of particular importance, because dysregula-tion of the normal balance between direct and indirectstriatal output neurons is thought to play a critical role inthe pathophysiology of movement disorders resulting frombasal ganglia dysfunction (Albin et al., 1989).

5-HT2C receptors in the subthalamic nucleus

The results of the present study have confirmed thepresence of a moderate-to-high level of expression of5-HT2C receptor mRNA in neurons of the subthalamicnucleus (Hoffman and Mezey, 1989; Mengod et al., 1990;Wright et al., 1995). Expression of 5-HT2C binding sitesand receptor mRNA within the subthalamic nucleus is ofinterest, because stimulation of 5-HT2C receptors withinthe subthalamic nucleus elicits orofacial dyskinesia in therat (Eberle-Wang et al., 1996a). Recent data support the

Figure 7 (Continued.)


Fig. 8. A: Low-power, darkfield photomicrograph of the substantianigra showing labeling of cells for 5-HT2C receptor mRNA (arrows). B:Low-power, brightfield photomicrograph of the same field as in Ashowing the distribution of TH mRNA-positive neurons (arrows).Stars in A and B mark the same artifact in the section. Note that thedistribution of neurons in A and B does not overlap; rather, clustered5-HT2C receptor mRNA-positive neurons are found in areas devoid of

TH mRNA-positive neurons. Inset: Enlargement of a TH mRNA-positive neuron stained with digoxigenin. C,D: High-power, bright-field photomicrographs of the substantia nigra showing that 5-HT2Creceptor mRNA-positive neurons (solid arrows) are often found nearTHmRNA-positive cells (open arrow), but THmRNA-positive neuronslack expression of 5-HT2C receptor mRNA (dark grains). Scale bars 550 µm inA,B, 20 µm in C,D, 10 µm in inset.


hypothesis that subthalamic 5-HT2C receptors are stimu-lated by endogenous 5-HT. Indeed, a lesion of the seroton-ergic inputs to the forebrain with 5,7-dihydroxytrypta-mine resulted in a marked supersensitivity of orofacialdyskinesia induced by the 5-HT2C agonist 1(m-chlorophe-nyl)-piperazine (m-CPP) and an increase in 5-HT2C mRNAlevels in the subthalamic nucleus (Eberle-Wang et al.,1996b).

Distribution of 5-HT2C receptor mRNAin the substantia nigra

The presence of neurons expressing the 5-HT2C receptormRNA in both pars compacta and pars reticulata of thesubstantia nigra is in agreement with previous reports(Hoffman and Mezey, 1989; Mengod et al., 1990; Wright etal., 1995), but the neurochemical identity of the cellsexpressing mRNA for 5-HT2C receptors had not beenpreviously documented. The pattern of expression of the5-HT2C receptor mRNAobserved in the substantia nigra inthis study presented two novel features. First, despite thepresence of labeled neurons in both pars compacta andpars reticulata, only those neurons that did not expressTH mRNA were positive for the 5-HT2C receptor mRNA.Second, not only did the density of labeled neurons varydramatically in rostral vs. caudal substantia nigra, but,even at the caudal levels, only a fraction of the neuronsexpressed detectable levels of labeling.Previous studies have reported a ‘‘spotty’’ labeling for

5-HT2C receptor mRNA in the substantia nigra pars com-pacta of rats (Mengod et al., 1990). In the present study,the cells within the substantia nigra expressing 5-HT2Creceptor mRNA were identified by using double in situhybridization histochemistry as expressing GAD, amarkerof GABAergic neurons, but not TH, amarker of dopaminer-gic neurons. It is unlikely that the absence of doublelabeling for TH and the 5-HT2C receptor mRNA wasrelated to a technical failure, because labeling for eachmRNAwas strong in the double-labeled slides, and double-labeled cells for GAD mRNA have been readily detectedwith the same method. Furthermore, the location and

density of neurons expressing the 5-HT2C receptor mRNAin the substantia nigra pars compacta corresponded tothose cells that did not express TH mRNA in single-labelexperiments (Mercugliano and Chesselet, unpublishedobservations).Although dopaminergic neurons constitute the large

majority of neurons in the pars compacta, this region alsocontains clusters of GABAergic neurons in the rat (Oertelet al., 1982; Chesselet et al., 1987). Nondopaminergicneurons in the pars compacta have been shown to projectto the striatal matrix (Gerfen et al., 1987b) and to thethalamus (Herkenham, 1979) or to send collaterals to bothof these regions (Deniau et al., 1978). Furthermore, GABA-ergic interneurons have been described in both pars com-pacta and the ventral tegmental area (Johnson and North,1992). Interestingly, stimulation of 5-HT2 receptors withthe nonselective 5-HT2A/C agonist (6) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; Leonhardt et al., 1992)exerts both direct effects on dopaminergic neurons andindirect effects through local GABAergic interneurons(Pessia et al., 1994). The selective expression of 5-HT2Creceptor mRNA in GABAergic neurons in the ventralmesencephalon suggests that these direct and indirecteffects are mediated by different subtypes of the 5-HT2receptor (Saudou and Hen, 1994). A functional role for5-HT2, possibly 5-HT2C, receptors expressed by GABAergicneurons in the substantia nigra is further supported byevidence that intranigral infusion of the mixed 5-HT2A/Cantagonist ritanserin blocks the decrease in nigral GABArelease induced by methylenedioxymethamphetamine(MDMA), an agent that releases 5-HT (Yamamoto et al.,1995). Behavioral studies in rats have shown that localadministration of the 5-HT receptor agonist m-CPP (Lim-inga et al., 1993) and of the 5-HT reuptake blockerfluoxetine (Pasini et al., 1992) into the substantia nigraaffect oral movements and seizure activity, respectively.Although it is nonselective, m-CPP acts at 5-HT2C recep-tors (Eberle-Wang et al., 1996a); therefore, its effects in thesubstantia nigra could be mediated by 5-HT2C receptorsthat are expressed by GABAergic neurons. However, thesestudies have not performed a detailed pharmacologicalcharacterization of these effects nor have they specificallyexamined whether or not these behavioral effects correlatewith the rostrocaudal location of the infusion within thesubstantia nigra.The anterocaudal differences in the density of neurons

expressing 5-HT2C receptor mRNA in the substantia nigrapars reticulata do not appear to be associated with differ-ences in the density of serotonergic innervation in thesubstantia nigra. Indeed, although previous immunohisto-chemical studies in rats (Steinbush, 1981) revealed ahigher density of serotonergic terminals in pars reticulatathan in pars compacta, they did not mention any markedheterogeneity in the distribution of serotonergic fibers atdifferent rostrocaudal levels of the substantia nigra. Thisobservation is supported by the presence of similar levelsof 3H-cyanoimipramine binding sites at the rostral andcaudal levels of the substantia nigra examined here.Accordingly, the actions of 5-HT may be mediated bydifferent receptor subtypes in different subregions of thesubstantia nigra. The presence of 5-HT1B, 5-HT1Db, 5-HT1Da,and 5-HT4 binding sites have all been reported in thesubstantia nigra (for review, see Saudou and Hen, 1994),but the specific function and detailed anatomical locationof these receptors are not yet fully known.

Fig. 9. High-power, brightfield photomicrograph of glutamic aciddecarboxylase (GAD) mRNA-positive neurons in the substantia nigrapars compacta. Two of the GAD mRNA-positive neurons, which aredarkly stained with digoxigenin, express 5-HT2C receptor mRNA(arrowheads), as revealed by the dark grains associated with each ofthe cell bodies. Arrow indicates a GAD-positive but 5-HT2C-negativeneuron. Scale bar 5 5 µm.


In addition to the anterocaudal gradient of 5-HT2CreceptormRNAexpression, only a subpopulation of GABA-ergic neurons in the substantia nigra pars reticulataexpressed this mRNA, even at caudal levels. Electrophysi-ological studies (Waszczak et al., 1984; Pessia et al., 1994)have revealed different responses of subpopulations ofGABAergic neurons in the pars compacta or the parsreticulata of the substantia nigra to dopaminergic andserotonergic agonists; however, little is known about theneurochemical differences underlying this response hetero-geneity. Expression of calbindin (CaBP28KDa) immunore-activity, a biochemical marker of matrix-associated meso-striatal projection neurons (Gerfen et al., 1987a), has aheterogeneous distribution within the substantia nigra,with high concentrations in the medial aspect of thesubstantia nigra pars reticulata in both rat (Gerfen et al.,1985; Celio, 1990) and human brain (Ito et al., 1992).However, there was no apparent correlation between thedistribution of cells expressing 5-HT2C receptor mRNAwith the distribution of calbindin-positive cells within thesubstantia nigra. The present data, to our knowledge,constitute the first evidence that marked neurochemicaldifferences exist among as-yet-unidentified subpopula-tions of GABAergic neurons in the substantia nigra.Elucidating these differences may prove to be critical for abetter understanding of the role of the substantia nigrapars reticulata in the control of movement.Efferent neurons of the substantia nigra pars reticulata

project mainly to the thalamus, the superior colliculus,and the tegmental area of the reticular formation (Beck-stead et al., 1979; Bentivoglio et al., 1979; Spann andGrofova, 1991; Deniau and Chevalier, 1992). Although theneurons of origin of these different projections have longbeen thought to be organized in topographically distinctclusters, recent data indicate a broad overlap in theirdistribution (Deniau and Chevalier, 1992). Because manypars reticulata neurons send collaterals to several of thesetargets in the rat (Deniau et al., 1978; Bentivoglio et al.,1979), subpopulations of neurons are more likely to becharacterized by a combination of outputs than by a singletarget area. The distribution of 5-HT2C receptor mRNA-expressing cells does not present any obvious similaritywith the lamellar distribution of neurons projecting todifferent sets of target structures (Deniau and Chevalier,1992). However, the much larger number of neuronsexpressing the 5-HT2C receptormRNAin the caudal part ofthe substantia nigra pars reticulata raises the possibilitythat this receptor may be preferentially associated withneurons projecting to the tegmentopontine nucleus, whichare more abundant in the caudal than in the rostral parsreticulata (Deniau and Chevalier, 1992). Because of thecomplexity of collateral projections of pars reticulata neu-rons (Deniau and Chevalier, 1992), a combination of tracingstudies with multiple tracers and in situ hybridization histo-chemistry will be necessary to further identify the GABAergicneurons that express the 5-HT2C receptor mRNA.

CONCLUSIONS

The death or dysfunction of dopaminergic neurons of thesubstantia nigra pars compacta leads to hypokinesia, asobserved in Parkinson’s disease. However, the GABAergicneurons of the substantia nigra, together with the efferentneurons of the internal pallidum, constitute the mainefferent pathways of the basal ganglia, and their dysfunc-tion is critical for the generation of both hypokinetic

(Parkinson’s disease) and hyperkinetic (chorea, hemiballis-mus) syndromes. Although electrophysiological as well asneurochemical data indicate an action of 5-HT on nigro-striatal neurons (Kelland et al., 1990; Pessia et al., 1994),the selective expression of the 5-HT2C receptor mRNA inGABAergic neurons, rather than dopaminergic neurons,in the caudal substantia nigra suggests that 5-HT affectsdifferent output neurons of the substantia nigra by actingon different subtypes of the serotonergic receptor. Togetherwith the selective expression of 5-HT2C receptor mRNA byneurons of the internal pallidum, and not the externalpallidum, this opens the possibility that pharmacologicalagents that act selectively on the 5-HT2C receptor mayhave selective effects on basal ganglia function, a hypoth-esis that could have critical implications for the treatmentof a variety of extrapyramidal movement disorders. In addi-tion, the observation that only a subpopulation of GABAergicneurons in the substantia nigra pars reticulata expressesdetectable levels of 5-HT2C receptor mRNA suggests anovel form of intrinsic organization within this region.

ACKNOWLEDGMENTS

We are grateful to Ms. Nicole Clavel for help with theexperiments; to Drs. Lewis, Chikaraishi, Tobin, and Pritch-ett for the gift of cDNAs; to Dr. H. Kung for the radioligand;and to Dr. S. Maayani for helpful discussions during thecourse of this study. This work was supported by PHSgrants MH-48125 and MH-44894 and by the TouretteSyndromeAssociation.

LITERATURE CITED

Albin, R.L., A.B. Young, and J. Penney (1989) The functional anatomy ofbasal ganglia disorders. Trends Neurosci. 12:366–375.

Altschul, S.F., W. Gish, W. Miller, E.W. Meyers, and D.J. Lipman (1990)Basic alignment search tool. J. Mol. Biol. 215:403–410.

Beckstead, R.M., V.B. Domesick, and W.J. Nauta (1979) Efferent connec-tions of the substantia nigra and ventral tegmental area in the rat.Brain Res. 175:191–217.

Bentivoglio, M., D. Van deer Caw, and H.G.J.M. Kuypers (1979) Theorganization of the efferent projections of the substantia nigra in therat. A retrograde fluorescent double labeling study. Brain Res. 174:1–17.

Berendse, N.W., H.J. Groenewegen, and A.H.M. Lohman (1992) Compart-mental distribution of ventral striatal neurons projecting to the mesen-cephalon of the rat. J. Neurosci. 12:2079–2103.

Celio, M.R. (1990) Calbindin D-28k and parvalbumin in the rat nervoussystem. Neuroscience 35:375–475.

Chesselet, M.-F. (1990) Appendix. In situ hybridization using radiolabeledRNA probes. In M.-F. Chesselet (ed): In Situ Hybridization Histochem-istry. Boca Raton, FL: CRC Press, pp. 189–203.

Chesselet, M.-F., and J.M. Delfs (1996) Basal ganglia and movementdisorders: An update. Trends Neurosci. 19:417–422.

Chesselet, M.-F., and L.-T. Weiss-Wunder (1994) Quantification of in situhybridization histochemistry. In J. Eberwine, K.L. Valentino, and J.D.Barchas (eds): In Situ Hybridization in Neurobiology. NewYork: OxfordUniversity Press, pp. 114–123.

Chesselet, M.-F., L. Weiss, C. Wuenschell, A.J. Tobin, and H.U. Affolter(1987) Comparative distributions of glutamic acid decarboxylase, tyro-sine hydroxylase, and tachykinins in the basal ganglia: An in situhybridization study in rodent brain. J. Comp. Neurol. 262:125–140.

Conn, P.J., E. Sanders-Bush, B.J. Hoffman, and P.R. Hartig (1986)Auniqueserotonin receptor in choroid plexus is linked to phosphatidylinositolturnover. Proc. Natl. Acad. Sci. USA 83:4086–4088.

Curzon, G., and G.A. Kennett (1990) m-CPP: A tool for studying behavioralresponses associated with 5-HT1C receptors. Trends Pharmacol. Sci.11:181–182.

Deniau, J.M., and G. Chevalier (1992) The lamellar organization of the ratsubstantia nigra pars reticulata: Distribution of projection neurons.Neuroscience 46:361–377.


Deniau, J.M., C. Hammond,A. Riszk, and J. Feger (1978) Electrophysiologi-cal properties of identified output neurons of the rat substantia nigra(pars compacta and pars reticulata): Evidence for the existence ofbranched neurons. Exp. Brain Res. 32:409–422.

Eberle-Wang, K., I. Lucki, and M.-F. Chesselet (1996a) A role for thesubthalamic nucleus in 5-HT2C-induced oral dyskinesia. Neuroscience72:117–128.

Eberle-Wang, K., A. Mehta, R.J.S. Banks, and M.-F. Chesselet (1996b)Regulation of 5-HT2C receptors in the subthalamic nucleus following5,7, DHT lesion in rats. FASEB J. 10:A714.

Erlander, M.G., and A.J. Tobin (1991) The structural and functionalheterogeneity of glutamic acid decarboxylase: A review. Neurochem.Res. 16:215–226.

Essman, W.D., P. McGonigle, and I. Lucki (1993)Anatomical differentiationwithin the nucleus accumbens of the locomotor stimulatory actions ofselective dopamine agonists and d-amphetamine. Psychopharmacology112:233–241.

Frazer, A., and J.G. Hensler (1994) Serotonin. In G. Siegel, B. Agranoff,R.W. Albers, and P. Molinoff (eds): Basic Neurochemistry, 5th Ed. NewYork: Raven Press, pp. 283–308.

Gerfen, C.R., K.G. Baimbridge, and J.J. Miller (1985) The neostriatalmosaic: Compartmental distribution of calcium binding protein andparvalbumin in the basal ganglia of the rat and monkey. Proc. Natl.Acad. Sci. USA 82:8780–8784.

Gerfen, C.R., K.G. Baimbridge, and J. Thibault (1987a) The neostriatalmosaic: III. Dissociation of patch-matrix mesostriatal systems. J.Neurosci. 7:3935–3944.

Gerfen, C.R., M. Herkenham, and J. Thibault (1987b) The neostriatalmosaic: II. Patch- and matrix-directed mesostriatal dopaminergic andnondopaminergic systems. J. Neurosci. 7:3915–3934.

Grima, B.,A. Lamouroux, F. Blanot, N. Faucon-Biguet, and J. Mallet (1985)Complete coding sequence of rat tyrosine hydroxylase mRNA. Proc.Natl. Acad. Sci. USA 82:617–621.

Harrington, M.A., P. Zhoung, S.J. Garlow, and D.C. Roland (1992) Molecu-lar biology of serotonin receptors. J. Clin. Psychiatr. 53:8–27.

Herkenham, M. (1979) The afferent and efferent connections of theventromedial thalamic nucleus in the rat. J. Comp. Neurol. 153:487–518.

Hoffman, B.J., and E. Mezey (1989) Distribution of serotonin 5-HT1C

receptor mRNA in adult rat brain. FEBS Lett. 247:453–462.Ito, H., S. Goto, S. Sakamoto, and A. Hirano (1992) Calbindin-D28k in the

basal ganglia of patients with parkinsonism. Ann. Neurol. 32:543–550.Jacobs, B.L., and C.A. Fornal (1993) 5-HT and motor control: A hypothesis.

Trends Neurosci. 16:346–352.Johnson, S.W., and R.A. North (1992) Opioids excite dopamine neurons by

hyperpolarization of local interneurons. J. Neurosci. 12:483–488.Jones, D.L., G.J. Mogenson, and M. Wu (1981) Injections of dopaminergic,

serotonergic and GABAergic drugs into the nucleus accumbens: Effectson locomotor activity in the rat. Neuropharmacology 20:29–37.

Julius, D., A.B. MacDermott, R. Axel, and T.M. Jessell (1988) Molecularcharacterization of a functional cDNA encoding the serotonin 1creceptor. Science 241:558–564.

Kelland, M.D., A.S. Freeman, and L.A. Chiodo (1990) Serotonergic afferentregulation of the basic physiology and pharmacological responsivenessof nigrostriatal dopamine neurons. J. Pharmacol. Exp. Ther. 253:803–811.

Kovachich, G.B., C.E. Aronson, D.J. Brunswick, and A. Frazer (1988)Quantitative autoradiography of serotonin uptake sites in rat brainusing [3H]cyanoimipramine. Brain Res. 454:78–88.

Lavoie, B., and A. Parent (1990) Immunohistochemical study of theserotonergic innervation of the basal ganglia of the squirrel monkey. J.Comp. Neurol. 299:1–16.

Leonhardt, S., E. Gorospe, B.J. Hoffman, and M. Teitler (1992) Molecularpharmacological differences in the interaction of serotonin with5-hydroxytryptamine1C and 5-hydroxytryptamine2 receptors.Mol. Phar-macol. 42:328–335.

Lewis, E.J., A.W. Tank, N. Weiner, and D.M. Chikaraishi (1983) Regulationof tyrosine hydroxylase mRNAby glucocorticoid and cyclic AMP in a ratpheochromocytoma cell line. J. Biol. Chem. 258:14632–14637.

Lewis, M.E., and F. Baldino (1990) Probes for in situ hybridizationhistochemistry. InM.-F. Chesselet (ed): In SituHybridizationHistochem-istry. Boca Raton, FL: CRC Press, pp. 1–21.

Liminga, U., A.E. Johnson, P.E. Andren, and L.M. Gunne (1993) Modula-tion of oral movements by intranigral 5-hydroxytryptamine receptoragonists in the rat. Pharmacol. Biochem. Behav. 46:427–433.

Lucki, I., H.R. Ward, and A. Frazer (1989) Effect of 1-(m-chlorophenyl)piperazine and 1-(m-trifluoromethyl) piperazine on locomotor activity.J. Pharmacol. Exp. Ther. 249:155–164.

Mengod, G., H. Nguyen, H. Le, C. Waeber, H. Lubbert, and J.M. Palacios(1990) The distribution and cellular localization of the serotonin 1Creceptor mRNA in the rodent brain examined by in situ hybridizationhistochemistry. Comparison with receptor binding distribution. Neuro-science 35:577–591.

Mercugliano, M., J.-J. Soghomonian, Y. Qin, H.Q. Nguyen, S. Feldblum,M.G. Erlander, A.J. Tobin, and M.-F. Chesselet (1992) Comparativedistribution of messenger RNAs encoding glutamic acid decarboxylase(Mr 65,000 and Mr 67,000) in the basal ganglia of the rat. J. Comp.Neurol. 262:245–254.

Numan, S., K.H. Lungren, D.E. Wright, J.P. Herman, and K.B. Seroogy(1995) Increased expression of 5-HT2 receptor mRNA in rat striatumfollowing 6-OHDA lesions of the adult nigrostriatal pathway. Mol.Brain. Res. 29:391–396.

Oertel, W.H., M.L. Tappaz, A. Berod, and E. Mugnaini (1982) Two-colorimmunocytochemistry for dopamine and GABAneurons in rat substan-tia nigra and zona incerta. Brain Res. Bull. 9:463–474.

Palacios, J.M., C. Waeber, C. Mengod, and M. Pompeiano (1991) Molecularneuroanatomy of 5-HT receptors. In J.R. Fozard and P.R. Saxena (eds):Serotonin: Molecular Biology, Receptors and Functional Effects. Basel:Birkhauser Verlag, pp. 5–20.

Parent, A., and L.-N. Hazrati (1995) Functional anatomy of the basalganglia. I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res.Rev. 20:91–127.

Pasini, A., A. Tortorella, and K. Gale (1992) Anticonvulsant effect ofintranigral fluoxetine. Brain Res. 593:287–290.

Paxinos, G., and C. Watson (1986) The Rat Brain in Stereotaxic Coordi-nates, 2nd Ed. San Diego: Academic Press.

Pazos, A., D. Hoyer, and J.M. Palacios (1984) The binding of serotonergicligands to the porcine choroid plexus: Characterization of a new type ofserotonin recognition site. Eur. J. Pharmacol. 106:539–542.

Pessia, M., Z.-G. Jiang, R.A. North, and S.W. Johnson (1994) Actions of5-hydroxytryptamine on ventral tegmental area neurons of the rat invitro. Brain Res. 654:324–330.

Saper, C.B. (1996) Any way you cut it: A new journal policy for the use ofunbiased counting methods. J. Comp. Neurol. 364:5.

Saudou, F., and R. Hen (1994) 5-Hydroxytryptamine receptor subtypes invertebrates and invertebrates. Neurochem. Int. 25:503–532.

Spann, B.M., and I. Grofova (1991) Nigropedunculopontine projection inthe rat: An anterograde tracing study with Phaseolus vulgaris-leucoagglutinin (PHA-L). J. Comp. Neurol. 311:375–388.

Steinbush, H.W.M. (1981) Distribution of serotonin-immunoreactivity inthe central nervous system of the rat—Cell bodies and terminals.Neuroscience 6:557–618.

Vaccarino, F.J., and J. Rankin (1989) Nucleus accumbens cholecystokinin(CCK) can either attenuate or potentiate amphetamine-induced locomo-tor activity: Evidence for rostral-caudal differences in accumbens CCKfunction. Behav. Neurosci. 103:831–836.

Van Bockstaele, E.J., A. Biscuas, and V.M. Pickel (1993) Topography ofserotonin neurons in the dorsal raphe nucleus that send axon collater-als to the prefrontal cortex and nucleus accumbens. Brain Res. 624:188–198.

Voorn, P., G.J. Docter, A.L. Jongen-Relo, and A.J. Jonker (1994) Rostrocau-dal subregional differences in the response of enkephalin, dynorphinand substance P synthesis in rat nucleus accumbens to dopaminedepletion. Eur. J. Neurosci. 6:486–496.

Waszczak, B.L., E.K. Lee, T. Ferraro, T.A. Hare, and J.R. Walters (1984)Single unit responses of substantia nigra pars reticulata neurons toapomorphine: Effects of striatal lesions and anesthesia. Brain Res.306:307–318.

Wolfe, J., M.S. Kreider, C. Goodman, and D.J. Brunswick (1987) Labeling invivo of serotonin uptake sites in rat brain after administration of[3H]cyanoimipramine. J. Pharmacol. Exp. Ther. 241:196–203.

Wright, D.E., K.B. Seroogy, K.H. Lundgren, B.M. Davis, and L. Jennes(1995) Comparative localization of serotonin 1A, 1C and 2 receptorsubtype mRNAs in rat b/rain. J. Comp. Neurol. 351:357–373.

Yamamoto, B.K., J.F. Nash, and G.A. Gudelsky (1995) Modulation ofmethylenedioxymethamphetamine-induced striatal dopamine releaseby the interaction between serotonin and gamma-aminobutyric acid inthe substantia nigra. J. Pharm. Exp. Ther. 273:1063–1070.

Zahm, D.S., and L. Heimer (1993) Specificity in the efferent projections ofthe nucleus accumbens in the rat: Comparison of the rostral poleprojection patterns with those of the core and shell. J. Comp. Neurol.327:220–232.


Pattern of expression of the serotonin2C receptor messenger RNA in the basal ganglia of adult rats

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