Dopamine Receptor Agonists Regulate Levels of the Serotonin 5=HT, Receptor and Its Mrna in a Subpopulation of Rat Striatal Neurons

The Journal of Neuroscience, June 1, 1996, 16(11):3727-3736

Dopamine Receptor Agonists Regulate Levels of the Serotonin 5=HT, Receptor and its mRNA in a Subpopulation of Rat Striatal Neurons

Nathalie Laprade,’ Fatiha Radja,* Tom& A. Reader,233 and Jean-Jacques Soghomonianl 1Centre de Recherche en Neurobiologie and Dkpat-tement d’Anatomie, Fault6 de Mgdecine, Universitb Lava/, Quebec, Canada, and Dkpartements de *Physiologic et 3Psychiatrie, Facultk de Mkdecine, Universit6 de Mont&al, Quebec, Canada

The effects of dopamine receptor agonists on the levels of the neurons after double-labeling of sections with a 5-HT,, and a striatal serotonin 5-HT,, receptor and its mRNA were investi- preproenkephalin (PPE) cRNA probe. In control unlesioned rats, gated in rats lesioned with 6-OHDA as neonates. The mRNA 5-HT,, mRNA labeling was distributed in PPE-labeled as well encoding for the .5-HT,, receptor was detected by in situ as in PPE-unlabeled striatal neurons. In 6-OHDA-lesioned rats, hybridization histochemistry and the binding to 5HT,, recep- increased 5-HT,, mRNA labeling was found only in PPE- tors was revealed with [‘251](2,5-dimethoxy-4-iodophenyl)2- unlabeled neurons and it was abolished after apomorphine or aminopropane ([‘251]DOI). In adult control unlesioned rats, la- SKF-38393 administration. These results demonstrate that beling with the 5-HT,, cRNA probe and with [‘251]DOI was agonists of dopamine receptors inhibit the expression of concentrated in medial sectors of the striatum. In 6-OHDA- 5-HT,, receptors in a subpopulation of presumed striato-nigral lesioned rats, labeling with the 5-HT,, cRNA probe or with neurons. We propose that this regulation plays an important [‘251]DOI was increased in the striatum, particularly in its lateral role in the control of motor activity by dopamine and 5-HT in the subdivisions. These increases were abolished after chronic basal ganglia. systemic administration of the dopamine receptor agonists apomorphine or SKF-38393. The mRNA levels encoding for the Key words: striatum; 5-HT; dopamine; 6-OHDA; 5-HT,, se- 5-HT,, receptor were further measured in individual striatal rotonin receptor; Dl dopamine receptor

Dopamine and 5-HT play an important role in several brain puts to the striatum is also evidenced in adult rats injected with functions. Altered regulation of these two nemotransmitters in 6-OHDA as neonates. These rats demonstrate an important de- the basal ganglia is associated with various behavioral dysfunc- crease in the number of dopaminergic afferents and, at the same tions including motor and obsessive-compulsive disorders time, an increased density of 5-HT axons in the striatum, predom- (Sandyk et al., 1988). The striatum, one of the major components inantly in its rostra1 half (Stachowiak et al., 1984; Berger et al., of the basal ganglia, receives a dense dopaminergic input from the 1985; Snyder et al., 1986; Descarries et al., 1992). The S-HT substantia nigra pars compacta as well as serotoninergic projec- hyperinnervation is paralleled by an increase in striatal 5-HT tions from the dorsal raphe nucleus. Serotoninergic and dopa- content and reuptake (Luthman et al., 1987; Molina-Holgado et minergic systems in the striatum interact with each other, and al., 1993, 1994). Neonatal 6-OHDA lesions also induce an in- such interaction might play a key role in their respective modes of creased ligand binding to striatal serotonin S-HT,,,, 5-HT,,,,,,,An, action. For instance, it has been demonstrated that 5-HT facili- and 5-HT,, receptors (Radja et al., 1993) and an increased tates the release of dopamine in the striatum (Benloucif et al., responsiveness of striatal neurons to iontophoretic application of 1993; Gallaway et al., 1993; Yadid et al., 1994; Bonhomme et al., 5-HT receptor agonists (El Mansari et al., 1994). An increase in 1995) and regulates the firing rate of dopamine neurons in the 5-HT,,, but not 5-HT,, or 5-HT,,- (also called S-HT,(.), mRNA substantia nigra (Kelland et al., 1990). Reciprocally, dopamine levels has also been recently reported in the striatum of rats afferents are able to facilitate the release of 5-HT in the raphe injected with 6-OHDA as adults (Numan et al., 1995). dorsalis and, at the same time, inhibit this release in the striatum Adult rats lesioned with 6-OHDA as neonates demonstrate (Lee and Geyer, 1984; Ferre and Artigas, 1993; FerrC et al., 1994). supersensitive behavioral responses to the administration of do- An interaction between dopaminergic and serotoninergic in- pamine Dl receptor agonists (Breese et al., 1985a,b). In addition, the motor hyperactivity exhibited by these rats under drug-free conditions has been shown to involve 5-HT,, receptors (Luthman Received Jan. 19, 1996; revised March 6, lYY6; accepted March 8, 1996. The studies were funded by the Parkinson Foundation of Canada, the Natural et al., 1991). These data indicate that both the Dl and 5-HT,, Sciences and Engineering Research Council, and the Fonds de Recherche en SantC receptor subtypes are preferentially involved in some of the motor du Quibec (FRSQ) to J.-J& and by the Medical Research Council of Canada abnormalities and adaptive changes exhibited by rats lesioned (MTl2966) and the FRSQ to T.A.R. We thank Dr. D. Pritchett for the gift of the 5-HTzA receptor cDNA and Ms. G. Audet and Ms. I. Deaudelin for their expert with 6-OHDA as neonates. In the present study, we tested the technical assistance. hypothesis that dopamine receptor agonists, particularly Dl ago- Correspondence should be addressed to Dr. Jean-Jacques Soghomonian, Centre nists, are involved in the modulation of striatal 5-HT2, receptor de Recherche en Neurobiologic, HBpital de I’Enfant-JCsus, 1401 18 Rut, Quebec, Canada GIJ 124. levels in adult rats lesioned with 6-OHDA as neonates. Because Copyright 0 1996 Society for Ncuroscicnce 0270.6474/96/163727-10$05.00/O striatal projection neurons can be distinguished as two subpopu- 3728 J. Neurosci., June 1, 1996, 16(11):3727-3736 Laprade et al. l 5-I-IT,, Receptor Regulation by Dopamine lations that either express or do not express the preproenkephalin and 50 mgiml X-phosphate (all these chemicals were purchased from (PPE) mRNA, we further analyzed the fate of the mRNA encod- Boehringer Mannheim). The reaction was stopped by dipping the slides in Tris buffer (10 mM; pH 8.0) containing I mM EDTA. Sections were ing for the SHT,, receptor in these two neuronal subpopulations. washed in 2X SSC for 15 min, quickly dipped in ammonium acetate (300 mM), rinsed in 70% ethanol, and air dried. Sections were first juxtaposed MATERIALS AND METHODS to Kodak X-OMAT-AR x-ray films for 21 d and then processed for emulsion radioautography. In that case, sections were dipped in the Neonu,ul 6-OHDA treatments. Three pregnant female Sprague-Dawley Amersham LM-1 nuclear emulsion, air dried, and stored at 4°C in rats (Charles River, Montreal) were housed individually with water and light-tight boxes in presence of desiccant. After 4-8 d of exposure, the dry food available ad libitum. Three days after delivery, each litter was emulsion radioautographs were developed in Kodak D-19 for 3.5 min at reduced to 12 pups. Twenty-five pups were given bilateral cerebroven- 14°C and mounted with Aquaperm mounting media (Fisher Scientific, tricular injections of the neurotoxin 6-OHDA (Sigma, St. Louis, MO) and Orangeburg, NY). 6 pups (sham-opcratcd) were injected with the vchiclc (0.9% sodium [“.‘I]DOI binding. The serotonin 5-HT ZA receptors were labeled with chloride and 1% ascorbic acid) under anesthesia with methoxyflurane [“‘I]DOI (DuPont, Billerica, MA, specific activity 2200 Ciimmol), ac- vapors. Pups were injected either with a total of 100 pg of 6-OHDA in 10 cording to Mengod’s modification (Mengod et al., 1990; Radja et al., ~1 (5 ~1 in each vcntriclc) or with 10 ~1 of vehicle (sham-operated). All 1993) of the protocol of McKenna et al. (1989). Briefly, the sections (20 animals were pretreated with the noradrenaline uptake inhibitor desipra- pm thick) wcrc preincubated at 25°C for 30 min in 50 nlM Tris-HCI buffer mine (25 mg/kg, s.c.) 45 min before surgery, in order to protect norad- (pH 7.4) containing 4 mM CaCl,, 0.1% ascorbic acid, and 0.1% bovine renergic neurons. serum. They were then incubated for 90 min in the same buffer containing Phurmacologicul treatments. Sixty days after the surgery, the sham- 200 PM [‘Z”I]DOl, in the presence of 30 nM of unlabeled 5-HT to block operated and six of the 6-OHDA-lcsioned rats were injected subcutaneously with vehicle (0.02% acetic acid in 0.9% NaCI). The other 6-OHDA- 5-HT,,. sites. Nonspecific binding was determined in the presence of 4 lesioned rats wcrc subdivided in three groups of six or seven animals that mM cold unlabeled 5-HT. After incubation with the radioligand, the slides were injected subcutaneously with the mixed dopaminc DI/D2 rcccptor were washed in cold butfer (2x 10 min) and dried under a stream of cold agonist apomorphinc (5 mg/kg), the preferential dopaminc DI receptor air. Autoradiographs wcrc gcneratcd by juxtaposition of the slides to agonist SKF-383Y3 (I 2.5 mgikg), or SKF-38393 in combination with the autoradiographic film (Hyperfilm, Amersham, Arlington Heights, IL), dopaminc Dl rcccptor antagonist SCH-23390 (0.2 mgikg). All injections together with Microscalcs (Amersham); the exposure lasted 3 d. were given twice daily for IO d. Three hours after the last injection, all rats /.‘H/r?zuzindo/ /Grzdir~g. The density of dopaminc rcuptakc sites in the were killed by decapitation; their brains were quickly removed and kept striatum was measured by [‘HI mazindol binding, as previously reported frozen at -70°C. Tissue sections (10 or 20 pm thick) wcrc cut at striatal by Javitch ct al., (1985). Frozen sections (IO Fm thick) were dried under level on a cryostat, thaw-mounted onto slides coated with gelatin, and a flow of air and rinsed for 5 min at 4°C in 50 mM Tris butfer with 120 mM stored at -70°C until further use. NaCl and 5 mM KCI to wash otf the endogenous l&and. They wcrc then Synthesis of the cKNA probes. Radioactive- or digoxigenin-labeled incubated for 40 min in 15 nM [‘Hlmazindol (DuPont NEN; specific cRNA probes were produced by in vitro transcription from cDNA clones activity, 22.7 Ciimmol) in 50 mM Tris buffer containing 300 mM NaCl and encoding for the rat S-HT2, receptor (Pritchctt et al., 1988) or the rat 5 mM KC]. Desipramine (0.3 pM) was added to the incubation medium to PPE (Yoshikawa et al., 1084). The cDNAs inserted into a PSP64165 block the norepinephrine transporter. Sections were then rinsed 2x 3 plasmid vector were linearized with EcoRI (5-HT,,) or SucI (PPE) min in the incubation buffer and 10 set in distilled water and then dried restriction enzymes. Transcription of the radioactive 5-HT,, cRNA under a flow of air at room temperature. Sections were then juxtaposed probe was performed using a riboprobe kit (Promega, Madison, WI) in to X-OMAT-AR x-ray films (Amersham) for 14 d. the presence of 2.5 pM [“S]UTP (1000 Ciimmol, DuPont NEN, Boston, /3H]cituloprum binding. The density of the striatal 5-HT innervation MA) and 10 pM unlabeled UTP. Transcription of the digoxigenin-labeled was estimated after citalopram binding to tissue sections. Fresh frozen PPE cRNA probe was performed in presence of 0.166 mM digoxigenin- brain sections (10 km thick) were dried and preincubated at room UTP (Boehr&ger Mannheim, Indianapolis, IN) and 0.33 mM-unlabeled temperature for 15 min in 50 mM Tris buffer (pH 7.4; containing 120 mM UTP. In both casts. unlabeled CTP. GTP. and ATP were added in excess. NaCl and 5 mM KC]). They were then incubated for 1 hr at room The reaction was ‘performed for 2 hr ‘at 37”C, and then the cDNA temperature in 1 nM [‘Hlcitalopram (DuPont NEN; specific activity, 82.0 templates were digested with the DNase I. The labeled cRNAs were Ci/mmol) diluted in the Tris buffer. Nonspecific binding was determined purified by phenol/chloroform extraction and ethanol precipitation. The by incubating control sections for 1 hr in the same solution containing 1 length of the cRNAs was reduced to 100-150 nucleotides by partial FM imipramine. After the incubation, the sections were rinsed 2X 10 min alkaline hydrolysis to improve accessibility of the probe (Cox et al., 1984). in ice-cold buffer and quickly dipped in ice-cold distilled water. They were In situ hybridizution and rudiouutogruphy. Brain sections (10 pm thick) then dried under a flow of cold air and juxtaposed for 3 weeks to were quickly dried at room temperature and fixed for 5 min in a solution tritium-sensitive films (Hyperfilms, Amersham). of 3% paraformaldehyde in phosphate buffer (I M; pH 7.2) containing Anulysis of the f&n radioautographs. The levels of radioautographic 0.02% DEPC. Sections were treated for 10 min with tricthanolamine (0.1 labeling on x-ray films were quantified in the striatum by computerized M, pH 8.0) containing 0.25% acetic anhydride and then for 30 min with densitometry with a Macintosh computer and an Ultimage image analysis Tris-glycinc (1 M, pH 7.0) before being dehydrated and air dried. Each software (Grdftck, France). The optical density of labcling in various section was covered with 3-3.5 ng of radiolabeled cRNA probe and 4 ng striatal sectors was calculated after subtracting the optical density of the of the digoxigcnin-labeled probe diluted in 20 ~1 of hybridization solution film and standardization against emulsion-coated filters (Kodak). Inter- (containing 40% formamide, 10% dextran sulfate, 4~ SSC, 10 mM nal 14C (for “‘S-labeled cRNA probe) or “‘1 (for “‘I-labeled DOI) dithiothrcitol, 1% sheared salmon sperm DNA, 1% yeast tRNA, IX standards (Amersham) were used to insure that measurements were Denhardt’s solution containing 1% RNase-free BSA). Some control made in the linear portion of the film. Labeling was measured in four sections were hybridized with a sense 5-HTzA or PPE RNA probe to different sectors in order to sample the whole striatal surface. Three veri@ the specificity of labeling. The sections were covered with Parafilm sections per animal were analyzed in each condition. The average level of and placed in humidified boxes, and hybridization was performed for 4 hr labeling was calculated for each rat and in each striatal sector sampled. at 50°C. Posthybridization washes were in 50% formamide (in 2X SSC) at Statistically significant difference in radioautographic labeling for each 52°C for 5 min and 20 min, in RNase A (100 pg/ml; Sigma; in 2X SSC) striatal sector in the different experimental groups of rats was determined for 30 min at 37”C, and in 50% formamide (in 2X SSC) at 52°C for 5 min. using a one-way ANOVA, whereas post-hoc paired comparisons were Sections were further rinsed at room temperature for 30 min in 2~ SSC performed with the PLSD Fisher’s test. Statistical significance was de- containing 0.05% Triton X-100 and for 3X 5 min in Tris buffer (0.1 M, pH fined asp < 0.05. 7.5) containing 0.15 M NaCl, 0.3% Triton X-100 and 2% normal sheep Analysis of emulsion radioautographs. The cellular distribution of the serum. Sections were then covered with 100 ~1 of an anti-digoxigenin Fdb 5-HT,,-receptor mRNA in striatal neurons expressing or not the PPE fragment conjugated with alkaline phosphatase (Boehringer Mannheim) mRNA was examined on emulsion radioautographs by light microscopy. diluted 1:500 in the same Tris buffer and left overnight at 4°C. Then, the First, all single or double-labeled neurons observed in one microscopic sections were rinsed for 3X 7 min in the antibody buffer and for 2X 5 min field were mapped on paper using a camera lucida. The mapping was in a Tris buffer (0. I M; pH 9.5) containing 0.1 M NaCl and 0.05 M MgCl. performed at a magnification of 25X and the microscopic field corre- The sections were then incubated in the dark for 2-5 hr in the same Tris sponded to an area of 0.212 mm’. From these maps, the numbers of butfer containing 0.24 mg/ml levamisole, 75 mg/ml nitroblue tetrazolium neurons labeled with the ““S-radioactive 5-HT,, cRNA probe alone or Laprade et al. l !%HT,, Receptor Regulation by Dopamine J. Neurosci., June 1, 1996, 76(11):3727-3736 3729 with the digoxigenin-labeled PPE cRNA probe were then calculated. In Table 1. Average numbers (per 0.212 mm’ of tissue section) of neurons order to provide an estimate of the ratio of labeled versus unlabeled labeled with the 5HT,, cRNA, double-labeled with the S-HT,, and neurons in each microscopic field, Nissl-stained neurons on adjacent PPE cRNAs, or Nissl-stained on adjacent sections in two sectors of the sections were similarly mapped. The levels of mRNA encoding for the striatum 5-HT,, receptor in individual neurons expressing or not the PPE mRNA was then measured on emulsion radioautographs under dark-field (for 5HTu (n) 5HT,,+PPE (n) Nissl PPE-labeled neurons) or bright-field (for PPE-unlabeled neurons) illu- mination at 40X magnification. The area covered by silver grains in each Medial Lateral Medial Lateral Medial Lateral neuron was measured by computerized image analysis (National Insti- Control 129212 llO?lO 66t5 6125 146 t 10 151 i 10 tutes of Health IMAGE 1.55) and expressed as a number of pixels per neuron. The number of pixels in each neuron was determined in an area Lesioned 16029 156%7* 8156 83t3* 152210 17Ok7 of constant dimension that was large enough to encircle the larger Apomorphine 13958 105 2 10 91 k4 6925 134 t 12 133 t- 19 neuronal profiles. A sample of -50 neurons labeled for PPE and 50 SKF-38393 139217 11929 80+8 7557 151 t 10 143 2 2 neurons unlabeled for PPE mRNA was thus analyzed for each rat. The average level of labeling from six rats in each experimental group was Average numbers of neurons sampled in a microscopic field corresponding to an area then calculated. Statistically significant differences in labeling for each of 0.212 mm*. The total number of neurons labeled with the 5-HTzA cRNA probe (5.HTzA), double-lab&d with the S-HT,, and the PPE cRNA probes (S- striatal sector between the experimental groups of rats were calculated HT,,+PPE), or Nissl-stained on adjacent sections was calculated in two sectors, with an ANOVA. Post hoc pairwise comparisons of 5-HT,, mRNA medial and lateral, of the striatum. Data are from adult control sham-operated rats labeling between experimental groups was performed for each striatal (control), rats that received h-OHDA as neonates (lesioned), and rats that received sector with a Fisher’s test with y < 0.05 considered significant. GOHDA as neonates and were treated with apomorphine or SKF-3X3% as adults. The values are means (?SEM) from six rats per condition. ANOVAs indicated significant differences between expcrimcntal groups fw the lateral striatal sector only (see Results). *, Significantly diffcrcnt from the controls or from the ;Ipomorphinc- RESULTS and SKF-.38.1Y.1-trcaled rats with the Fisher’s test. Regional and cellular distribution of the 5-HT,, receptor and its mRNA in control rats Effect of neonatal 6-OHDA injections and Labeling with the 5-HT,, cRNA probe or with [‘2’1]DOI exhib- administration of dopamine-receptor agonists on ited a latero-medial gradient of distribution in the striatum of 5-HT,, mRNA levels control rats (Fig. IA,@. Quantitative analysis of the film radio- Brain sections from adult, sham-operated or 6-OHDA-lcsioned, autographs demonstrated that the labeling with the 5-HT,, rats were first processed for [‘Hlmazindol binding to evaluate the cRNA probe was higher (+33% on average) in the medial than in loss of dopamine axon terminals in the striatum after neonatal the lateral sectors of the striatum (0.048 t 0.002 vs 0.032 i 0.002; 6-OHDA injections. Intense [‘Hlmazindol labeling was observed n = 6). Similarly, the level of [‘*“I]DOI binding was higher in the striatum of sham-operated rats (Fig. 24). In contrast, very (+43% on average) in the medial than in the lateral striatal weak labeling was observed in the striatum of rats injected with sectors (0.136 t- 0.023 vs 0.078 5 0.012; n = 6). 6-OHDA as neonates and treated or not with apomorphine or Observation of the emulsion radioautographs revealed the SKF-38393 as adults (Figs. 2B, 3). In accordance with a previous presence of neurons intensely labeled with the dark-blue alkaline- report (Molina-Holgado et al., 1994), the levels of [‘Hlcitalopram phosphatase reaction product in the striatum. In contrast, no such binding to 5-HT reuptake sites were significantly increased in the labeling could be detected in the overlying cerebral cortex (not striatum of rats injected with 6-OHDA as neonates (Fig. 2C,D). shown). In many instances, the digoxigenin-unlabeled neuronal Chronic administration of apomorphine or SKF-38393 to these profiles could be distinguished from the surrounding neuropil as rats did not affect the levels of [3H]citalopram binding that re- light blue spots (see Fig. 5). In addition, the presence of silver mained significantly higher than the levels measured in sham- grains accumulations on digoxigenin-unlabeled neurons allowed operated rats (Fig. 3). The quantification of labeling with the 5-HT2, cRNA probe their unambiguous identification. Neuronal profiles showing an was performed on x-ray film radioautographs at two frontal levels accumulation of three or more silver grains were considered of the striatum; that is, A = 10 and A = 9.2, according to the labeled with the S-HT,, cRNA probe. stereotaxic atlas of Paxinos and Watson (1986). At the rostral- Labeling with the 5-HT,, cRNA probe was visible in most level (A = lO.O), the ANOVAs demonstrated significant digoxigenin-labeled as well as in digoxigenin-unlabeled neurons. differences in 5-HT,, mRNA levels between the five experimen- Comparison with adjacent Nissl-stained sections indicated that tal groups of rats in the dorsomedial (Fc4,23) = 7.4; p = 0.0006), almost 90% of the striatal neurons in the medial and -70% of the the ventromedial (F(,,,,) = 6.6; p = O.OOll), the dorsolateral striatal neurons in the lateral sector were labeled with the 5-HT,, (Fc4,23) = 5.8, p = 0.0022), and the ventrolateral (Fcd,2.1) = 5.9, p cRNA probe (Table 1). The average numbers of neurons labeled = 0.0021) striatal sectors. At the caudal-most level (A = 9.2), the with the S-HT,, cRNA probe (PPE-labeled and PPE-unlabeled) ANOVAs demonstrated significant differences in 5-HT,, mRNA were slightly lower but not significantly different in the lateral than levels between the five experimental groups in the dorsomedial in the medial striatal sector (Table 1). A large majority (over (Fpwj = 8.6, p = 0.0002), the dorsolateral (Fc4,2sj = 8.5, p = 95%) of PPE-labeled neurons exhibited 5-HT,, labeling in the 0.0002), the ventrolateral (Fc4,25j = 9.0, p = 0.0001) but not the lateral and medial striatal sectors. In both striatal sectors, a ventromedial striatal sector. proportion of SO-60% of neurons expressing the 5-HT,, mRNA At the rostral-most level of the striatum, the average level of was also labeled with the PPE cRNA probe. As estimated from labeling with the 5-HT,, cRNA probe was significantly increased one representative control rat, the level of labeling with the in the four striatal sectors of 6-OHDA-lesioned rats when com- 5-HT,, cRNA probe was higher in neurons of the ventromedial pared to the labeling in sham-operated rats (Fig. 4A). The in- than in neurons of the ventrolateral striatal sector in both PPE- creased labeling was more prominent in the lateral than in the unlabeled (+48%; 100.2 i 7.7 vs 67.9 5 4.2 pixels per neuron; n medial striatal sectors (Fig. 4A). At the caudal-most level of the = 50) and PPE-labeled (+35%; 107.6 ? 8.4 vs 79.7 2 7.2 pixels striatum, the average 5-HT,, mRNA labeling was increased in per neuron; II = 50) neurons. the dorsolateral, the ventrolateral, and the dorsomedial sectors of 3730 J. Neurosci., June 1, 1996, 16(11):3727-3736 Laprade et al. l 5-HT,, Receptor Regulation by Dopamine

Figure 1. Negative images of x-ray films from frontal brain sections processed for in situ hybridization with a 35S-1abe1ed5-HT,, cRNA probe (exposure time 10 d;A, C, E, G) or incubated with [‘251]DOI (exposure time 3 d; B, D, F, H>. Sections are from adult control sham-operated rats (A, B), adult rats injected with 6-OHDA as neonates (C, D), and from adult rats injected with 6-OHDA as neonates and treated chronically with apomorphine (E, F) or SKF-38393 (G, H). The labeling intensity in each condition was measured by computerized densitometry in the four striatal sectors illustrated in Figure 1B. The surface of analysis shown for the dorsomedial sector was identical for the three other striatal sectors. Laprade et al. l 5-HTzA Receptor Regulation by Dopamine J. Neurosci., June 1, 1996, 16(11):3727-3736 3731

Figure 2. Negative images of x-ray films from frontal brain sections processed for [3H]mazindol (exposure time 3 weeks;% B) or [3H]citalopram (exposure 4 weeks; C, D) binding. Sections are from adult control sham-operated rats (A, C) or adult rats injected with 6-OHDA as neonates (B, D).

6-OHDA-lesioned rats (Fig. 4B). At the two frontal levelsexam- dorsolateral and the ventrolateral striatal sectorsonly (Figs. 1D ined, the labeling with the. 5-HT,, cRNA probe in 6-OHDA- 4C). In the medial striatal sectors,there were small increasesin lesionedrats appearedhomogeneously distributed over the whole labeling that did not reach statisticalsignificance (Fig. 4C). As a striatal surface (Fig. 1C). Chronic apomorphineor SW-38393 result of the preferential increasein the lateral striatal sectors, administrationto adult rats lesionedwith 6-OHDA as neonates [‘251]DOI labeling in 6-OHDA-lesioned rats appearedhomoge- abolishedthe increasesin 5-HT,, mRNA levelsin all, except the neously distributed over the whole striatal surface (Fig. 1D). ventromedial, striatal sectors(Figs. lC,E,G, U,B). As a conse- Chronic administration of apomorphine or SKF-38393 to quence,the striatum of rats lesionedwith 6-OHDA as neonates 6-OHDA-lesioned rats abolishedthe increasesin [‘251]DOI bind- and treated with apomorphineor SKF-38393 exhibited a pro- ing levelsin the dorsolateraland ventrolateral striatal sectorsbut nounced latero-medial gradient of labeling with the 5-HT,, did not produce any statistically significanteffect in the dorsome- cRNA probe that resembledthe gradient observed in sham- dial or ventromedial striatal sectors (Figs. lD,F,H, 4C). The operatedrats (Fig. l&G). The effects of SKI?-38393on 5-HT,, selective effect of apomorphine and SKF-38393 in the lateral mRNA levelswere blocked by concomitant administrationof the striatal sectorsresulted in a pronouncedlatero-medial gradient of dopamineDl receptor antagonistSCH-23390 (Figs. 4/&B). distribution of [1251]DOIlabeling that resembledthe distribution observedin sham-operatedrats (Fig. lF,H). The effectsof SKF- Effects of neonatal 6-OHDA injections and 38393on striatal [1251]DOIlevels were antagonizedby the con- administration of dopamine receptor agonists on [‘251]DOI binding levels comitant administrationof SCH-23390(Fig. 4C). [12sI]DOI binding levelswere measuredon x-ray film radioauto- Cellular distribution of the mRNA encoding for the graphsat only one frontal level of the striatum (A = 9.2). The 5-HT, receptor ANOVAs performed for each striatal sector revealed highly sig- Analysis of the emulsionradioautographs indicated that, as in nificant differencesin [12sI]DOI binding levels between experi- control rats, the 5-HT,, mRNA labeling in 6-OHDA-lesioned mental groupsin the dorsomedial(Fc4,21j = 5.2,~ = 0.0047),the rats was distributed in PPE-labeledas well as in PPE-unlabeled dorsolateral (F(,,,,) = 10.8,p < O.OOOl),and the ventrolateral neurons (Table 1). In addition, more than 95% of PPE-labeled (Fvs,,,) = 9.2,~ = 0.002)but not the ventromedial (Fc,,,,) = 2.8, neuronsalso expressed the 5-HT,, mRNA. In eachexperimental p = 0.0507)striatal sector. group, the numbersof neuronsexclusively labeled with the radio- When comparedto sham-operatedrats, [ ‘251]DOI binding lev- active 5-HT, cRNA probe or double-labeledwith the 5-HT,, els in 6-OHDA-lesioned rats were significantly increasedin the and the PPE cRNA probeswere not significantlydifferent in the 3732 J. Neurosci., June 1, 1996, 76(11):3727-3736 Laprade et al. l 5-HT,, Receptor Regulation by Dopamine

600 q Control I4 Control q Lesioned Apomorphine In 500 q SKF-38393 q SKF-38393 E tii SKF-38393 + SCH-23390 0 SKF-38393+SCH-23390 zx 400 ~-HT~A mRNA levels IA=1 0.01 t A g 300 500 ** If 2 z 400 e 200

2 300 100 200 0 [3H IMarindol [3HlCitalopram 100

Figure 3. Level of [“Hlmazindol or [3H]citalopram binding in the stria- turn of adult control sham-operated rats (control), adult rats lesioned with 0 6-OHDA as neonates (lesioned), and adult rats injected with 6-OHDA as DM VM DL VL neonates and chronically injected with apomorphine, SKF-38393, or a combination of SKF-38393 and SCH-23390. Labeling was measured by ~-HT~A mRNA levels {A=9.2) computerized densitometry on x-ray film radioautographs. The values ** represent the average labeling from six rats in each experimental group and are expressed as a percentage of the controls. ANOVAs for [‘Hlmazindol or [3H]citalopram binding indicated statistical significant differences between experimental groups (Fc4,241 = 4.5, p < 0.0001 and F(4.24) = 3.1, p < 0.05, respectively). *p < 0.05, **I, < 0.005 when compared to controls with the Fisher’s test.

lateral and medial striatal sectors (Table 1). In addition, the numbers of single- or double-labeled neurons in the medial striatum were not significantly different between experimental groups (Table 1). In the lateral striatal sector, however, the ANOVAs DM VM DL VL indicated a significant difference between experimental groups in [12511DOl binding levels (A=9.2) the number of neurons expressing the 5-HT,, mRNA (Fc4,23j = 11 11 3.5; p = 0.0231) or expressing both the 5-HT,, and the PPE 400 = ** ** 1 T T mRNAs (Fc4,23j = 4.6; p = 0.0073). Therefore, the numbers of neurons (labeled or not with the PPE cRNA probe) expressing the 300 5-HT,, mRNA were slightly higher in 6-OHDA-lesioned rats when compared to the sham-operated rats or when compared to the 6-OHDA-lesioned rats that were treated with apomorphine or 200 SKF-38393 (Table 1). In contrast, the numbers of Nissl-stained neuronal profiles in all these groups were not significantly different (Table 1). This indicated that some striatal neurons in the 100 lateral sector of control and 6-OHDA-lesioned rats treated with apomorphine or SKF-38393 did not express the 5-HT,, mRNA 0 or were below the threshold of detection. DM VM DL VL Quantification of 5-HT,, mRNA levels was then performed on emulsion radioautographs in individual neurons labeled or unlabeled Figure 4. Levels of 5-HT,, mRNA (A, B) or [““I]DOI binding (C) in with PPE in a ventrolateral striatal sector (Fig. 5). The ANOVAs the striatum of adult control sham-operated rats (control), adult rats indicated significant differences between experimental groups in the lesioned with 6-OHDA as neonates (lesioned), and adult rats injected with 6-OHDA as neonates and chronically injected with apomorphine, number of pixels per neuron in PPE-unlabeled (Fc4,2,jj = 6.4, p = SKF-38393, or a combination of SKF-38393 and SCH-23390. The 0.0018) but not PPE-labeled (Fc4,2,jj = 0.406, p = 0.8020) striatal values represent the average intensity of labeling measured on x-ray neurons (Fig. 6). Pairwise comparisons with sham-operated rats films by computerized densitometry and expressed as a percentage of showed that the 5-HT, mRNA labeling in 6-OHDA-lesioned rats the controls at frontal levels A = 10 or A = 9.2 according to the stereotaxic atlas of Paxinos and Watson (1986). The data (mean 2 was significantly increased in PPE-unlabeled neurons (Figs. 5A,B, 6). SEM) were obtained from six rats in each group. Labeling was mea- This increase was abolished after apomorphine or SIG38393 ad- sured in four different striatal sectors (DM, dorsomedial; VM, ventro- ministration (Figs. 5B-D, 6). The effect of SIG-38393 on 5-I-IT,, medial; DL, dorsolateral; and VL, ventrolateral). Statistical differences mRNA labeling in PPE-unlabeled neurons was blocked by concom- in labeling in each striatal sector were determined after a one-way itant administration of SCH-23390 (Fig. 6). The histograms of fre- ANOVA. Pairwise comparisons between different experimental conditions were made according to the Fisher’s test. *, p < 0.01, **, p < quency distribution of the 5-HT,, mRNA labeling in PPE-labeled 0.005 when compared to the controls; #, p < 0.01, or ##, p < 0.005 and PPE-unlabeled neurons shown in Figure 7 illustrate the increase when compared to the 6-OHDA-lesioned; and 8, p < 0.01, or Ylq, p < of 5-HT,, mRNA labeling in the population of PPE-unlabeled 0.005 when compared to the SKF-38393-treated rats. Laprade et al. . SHT,, Receptor Regulation by Dopamine J. Neurosci., June 1, 1996, 76(11):3727-3736 3733

Figure 5. Bright-fieldphotomicrographs of brain sections processed for in situhybridization histochemistry with a 35S-labeled5-HT,, cRNA probeand a digoxigenin-labeledPPE cRNA probein a ventrolateralstriatal sector. Labeling is from an adultcontrol sham-operated rat (A), an adultrat lesioned with 6-OHDA asneonate (B), andan adultrat lesionedwith 6-OHDA asneonate and chronically injected with apomorphine(C) or SW-38393(0). Neuronslabeled with the 5-HT,, cRNA probe are indicatedby the UTYOWS.Note the increasedlabeling on the PPE-unlabeledneuron of the 6-OHDA-lesionedrat (B). Scalebar, 10pm. neuronsin 6-OHDA-lesionedrats and its reversalafter apomorphine consistentwith previousreports (Fishette et al., 1988;Mengod et or SKF-38393administration. al., 1990;Pompeiano et al., 1994).A proportion of striata15-HT,, receptorswould also be localized on dopaminergicnerve termi- DISCUSSION nals (Muramatsu et al., 1988).However, this fraction of receptors Our resultsindicate that neonatal 6-OHDA lesionsinduce con- was probably not detected in 6-OHDA-lesioned rats, and the comitant increasesin the levelsof serotonin5-HT,, receptor and changesin striatal [‘2sI]DOI binding levelsmeasured in theserats mRNA in the adult rat striatum. Such increasesare abolishedin most likely reflect changes in the number of postsynaptic the lateral sectors of the striatum after chronic and systemic receptors. administrationof apomorphineor SKF-38393. The changesin After neonatal6-OHDA lesions,increased levels of the 5-HT,, mRNA levelsencoding for the .5-HT,, receptor are restricted to receptor and mRNA were particularly prominent in the lateral a subpopulationof striatal neuronsthat do not expressthe PPE striatal sectors.As a consequence,the heterogeneousdistribution mRNA. of labeling observedin control rats becamerather homogeneous Distribution of the striatal 5-HT, receptor and in 6-OHDA-lesioned rats. Chronic administration of apomor- its mRNA phine or SKI?-38393resulted again in a pronouncedlatero-medial The distribution of labelingwith the 5-HT,, cRNA probe in the gradient of distribution of labeling. Cellular analysisindicated that striatum of control rats was similar to the distribution observed this gradient wasprimarily attributable to higher 5-HT,, mRNA with [‘2sI]DOI. In both cases,labeling was heterogeneous and was levels in neuronsof the medial striatal sectors.Altogether, these more intensein the medial sectorsof the striatum. This similar resultssuggest that the heterogeneousdistribution of the 5-HT,, distribution of labelingis a strongindication that the cRNA probe receptor in the rat striatum is under the control of dopamine.In and [12sI]DOI specificallylabeled the 5-HT, mRNA and recep- particular, dopamineappears to exert an inhibitory control on the tor, respectively.This is consistentwith previous reports showing expressionof the 5-HT,, receptor and/or mRNA in neuronsof that DO1 in presenceof 30 nM 5-HT labelsthe 5-HT,, but not the the lateral striatum. closelyrelated 5-HT,, (formerly 5-HT,,) receptor site (Mengod At the caudal-mostlevel examined,the correspondencebetween et al., 1990). The comparabledistribution of labeling with the the levelsof 5-HT2* mRNA and[‘2sI]DOI bindingwas not observed cRNA probe and with [12sI]DOI also suggeststhat most striatal in the dorsomedialsector of the striatum.In this sector,increased 5-HT,, receptorsare distributedin cell bodies.This conclusionis 5-HT, mRNA levelsin 6-OHDA-lesionedrats were paralleled by a 3734 J. Neurosci., June 1, 1996, 76(11):3727-3736 Laprade et al. l SHT,, Receptor Regulation by Dopamine

5-HTZA mRNA levels per neuron that do not express the PPE mRNA. It has been previously shown that the majority of striato-pallidal neurons contain the mRNA El Control encoding for enkephalin whereas the majority of striato-nigral Lesioned w neurons express the mRNAs encoding for dynorphin and sub- * Apomorphine q SKF-38393 stance P, but not enkephalin (Gerfen et al., 1990; for review, see + 200 q SKF-38393+SCH-23390 also Gerfen, 1992). Thus, our results suggest that the 5-HT,, L T r mRNA is expressed in both striato-pallidal and striato-nigral neurons but its regulation by dopamine receptors occurs only in E 150 0 striato-nigral neurons. Striato-nigral neurons have been shown to 8 preferentially express the dopamine Dl receptor (Gerfen et al., dE 100 1990) whereas striato-pallidal neurons express the D2 receptor (Gerfen et al., 1990; Le Moine et al., 1990). It can thus be t speculated that Dl receptors are coupled to intracellular path- g 50 ways that directly participate in the regulation of the 5-HT,, receptor and/or mRNA.

0 PPE-unlabeled PPE-labeled Functional consequences of SHT, receptor regulation Figure 6. Levels of S-HT,, mRNA labeling in single PPE-labeled and The increased number of 5-HT,, receptors after neonatal PPE-unlabeled neurons in a ventrolateral sector of the striatum. Radio- 6-OHDA injections may result in hypersensitive responses of autographic labeling was measured by computerized image analysis (see Materials and Methods for details). The values are means 5 SEM of the striatal neurons to serotonin. This interpretation is supported by average number of pixels per neuron and arc cxprcsscd as a pcrccntagc of previous findings of increased responsiveness of striatal neurons the controls. Data are from adult control sham-opcratcd rats (control), to the inhibitory action of 5-HT or DOI (El Mansari et al., 1994). rats that rcceivcd 6-OHDA as neonates (lesioned), and rats that received Another study has shown, however, that 5-HT in such rats elicit 6-OHDA as neonates and were treated with apomorphinc, SKF-38393, or excitations rather than inhibitions of striatal neurons (Luthman et a combination of SKF-38393 and SCH-23390 as adults. A sample of 50 neurons per rat from six rats per experimental condition was analyzed. al., 1993). Eventual changes in the responsiveness of striatal Pairwise comparisons between experimental groups wcrc made with a neurons to 5-HT,, receptor agonists after neonatal 6-OHDA Fisher’s test. *, p < 0.01 when compared to the controls; #,p < 0.01 when would be associated with an increase in evoked release of striatal compared to the lesioned rats; and 7, p < 0.01 when compared to the 5-HT (Jackson and Abercrombie, 1992) without concomitant SKF-38393-treated rats. changes in the extracellular levels or basal release of 5-HT (Jack- son and Abercrombie, 1992; Luthmann et al., 1993; Molina- small but nonsignificant increase in [‘*‘I]DOI binding levels. In Holgado et al., 1993, 1994). After chronic administration of do- addition, administration of apomorphine or SKF-38393 abolished pamine receptor agonists to rats lesioned with 6-OHDA as the increased levels of the S-HT,, mRNA, but it had no consistent neonates, it can be expected that the hypersensitivity of striatal effect on [‘2’I]DOI binding levels. This suggests a certain degree of neurons to 5-HT receptor agonists will be reversed or attenuated mismatch between the regulation of the mRNA and the receptor as a consequence of decreased expression of the 5-HT,, receptor. itself in this dorsomedial striatal sector. Previous reports have shown that systemic administration of Regulation of striatal 5-HT receptors by dopamine DO1 to adult rats can induce an increase in striatal substance P receptor agonists mRNA and peptide levels (Walker et al., 1991). In addition, Administration of apomorphine or SKF-38393 had a comparable lesions of 5-HT neurons with 5,7-dihydroxytryptamine result in a inhibitory effect on the levels of the striatal 5-HT,, receptor and decrease in dynorphin levels without concomitant changes in the its mRNA. Furthermore, the effect of SKF38393 was blocked by levels of striatal PPE mRNA (Morris et al., 1992). On the other the preferential dopamine Dl receptor antagonist SCH-23390. hand, a facilitator-y role of Dl receptor agonists on the levels of These results strongly suggest that the effects of apomorphine and striatal dynorphin and substance P mRNAs has been documented SKF-38393 are mediated by Dl receptors. In normal rats, systemic previously (Gerfen et al., 1990). Altogether, these studies indicate administration of apomorphine has been shown to induce an that dopamine through Dl receptors, and 5-HT through 5-HT,, increase in the intracellular levels of 5-HT in the raphe dorsalis receptors, exert a facilitatory control on the expression of peptides and a decrease in the extracellular concentration of 5-HT in the in striato-nigral neurons. It is therefore possible that the control of striatum (Lee and Geyer, 1992; FerrC et al., 1994). The regulation dopamine Dl receptors on the expression of serotonin 5-HTzA of 5-HT levels by apomorphine is mediated by dopamine D2, but receptors has important consequences on the regulation of neu- not Dl, receptors in the raphe dorsalis (Ferre and Artigas, 1993). rotransmitters in striato-nigral neurons. In addition, when directly infused into the striatum, apomorphine Adult rats injected with 6-OHDA as neonates do not exhibit the or SKF-38393 do not alter the extracellular concentration of severe behavioral abnormalities observed when similar extensive serotonin (Fern? et al., 1994). In light of these previous and our lesions are performed on adults (Breese et al., 1984, Bruno et al., own results, it seems unlikely that the effects of apomorphine or 1987; Weihmuller and Bruno; 1989; Zigmond et al., 1990; John- SKF-38393 on the levels of the 5-HT,, receptor and its mRNA son and Bruno, 1992). However, these rats exhibit some learning involve an action on striatal 5-HT neurons. This interpretation is deficits as well as a motor hyperactivity and a behavioral hyper- also supported by the fact that apomorphine or SKF-38393 failed sensitivity to the administration of dopamine Dl agonists (Erinoff to alter the increases in citalopram binding levels measured in et al., 1979; Heffner et Seiden, 1982; Breese et al., 1984, 1985a,b; 6-OHDA-lesioned rats. Schallert et al., 1989; Gong et al., 1992, 1993). The motor hyper- Changes in 5-HT,, mRNA levels in 6-OHDA-lesioned rats activity can be reversed by the systemic administration of ketan- were exclusively observed in the subpopulation of striatal neurons serin or mianserin and therefore appears to be mediated by Laprade et al. . SHT,, Receptor Regulatm by Dopamine J. Neurosci., June 1, 1996, 76(11):3727-3736 3735

PPE-unlabeled nmrmm PPE-labeled neurons

A 16 3 c#rltml

16 12 8 4 0

16 12 8 F&z 7. Histograms of frequency dis- 4 tributions of 5-HT,, mRNA labeling in 0 PPE-labeled and PPE-unlabeled neurons of the lateral striatum. Data are from adult control sham-operated rats (Con- trol), rats that received 6-OHDA as neonates (Lesioned), and rats that received 16 16 6-OHDA as neonates and were treated with apomorphine, SKF-38393 or a com- 12 12 bination of SKF-38393 and SCH-23390 as adults. Quantification of silver grains 8 8 over individual striatal neurons was performed by computerized image analysis 4 4 (see Materials and Methods for details). 0 The area covered by silver grains is expressed in number of pixels per neuron. A sampie of 50 neurons per rat from six rats in each experimental condition was Number of pixels per neuron analyzed.

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