Serotonin 5-HT2A but Not 5-HT2C Receptor Antagonism Reduces

Neuropharmacology 49 (2005) 350e358 www.elsevier.com/locate/neuropharm

Serotonin 5-HT2A but not 5-HT2C receptor antagonism reduces hyperlocomotor activity induced in dopamine-depleted rats by striatal administration of the D1 agonist SKF 82958

Christopher Bishop, Gregory S. Daut, Paul D. Walker *

Department of Anatomy & Cell Biology, Wayne State University School of Medicine, 540 East Canﬁeld, Detroit, MI 48201, USA

Received 5 August 2004; received in revised form 7 March 2005; accepted 18 March 2005

Abstract

While recent work has indicated that D1 receptor agonist-induced hyperlocomotion in DA-depleted rats is reduced by striatal 5-HT2 receptor antagonism, the 5-HT receptor(s) subtypes mediating these effects are not yet known. In the present study, we examined the influence(s) of striatal 5-HT2A and 5-HT2C receptors on locomotor behavior induced by D1 agonism in neonatal DA- depleted rats. On postnatal day 3, male SpragueeDawley rats (n Z 68) were treated with either vehicle or 6-hydroxydopamine (6-OHDA; 60 mg) which produced O98% DA depletion. Sixty days later, all rats were fitted with bilateral striatal cannulae. A subset of control and 6-OHDA-lesioned rats (n Z 20) was tested for locomotor responses to striatal infusion of the D1 agonist SKF 82958 (0, 0.1, 1.0, 10 mg/side). The remaining rats (n Z 48) were tested for locomotor responses to intrastriatal SKF 82958 (2.0 mg/ side) alone or in combination with the 5-HT2A- or 5-HT2C-preferring antagonists M100907 or RS102221 (0.1 or 1.0 mg/side), respectively. Intrastriatal SKF 82958 dose-dependently increased measures of motor activity within DA-depleted rats. This hyperlocomotor activity was suppressed by co-infusion of M100907, but not RS102221. These results indicate that DA depletion strengthens striatal 5-HT2A/D1 receptor interactions and suggest that 5-HT2A receptor antagonists may prove useful in reducing D1-related movements. Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: 6-Hydroxydopamine; Basal ganglia; Dopamine; Locomotion; Parkinson’s disease; Striatum

1. Introduction (Kostrzewa et al., 1998; Reader and Dewar, 1999). While these compensatory mechanisms may initially Selective destruction of central dopamine (DA) mask underlying motor deﬁciencies that arise as a result neurons with the catecholaminergic neurotoxin 6- of altered striatal output, challenge with DA and 5-HT hydroxydopamine (6-OHDA) produces neuroplasticity receptor agonists can produce exaggerated motor within the striatum, altering the regulation and responses. For instance, D1 receptor stimulation within function of DA and serotonin (5-HT) systems the DA-depleted striatum of rats signiﬁcantly enhances locomotor activity (Breese et al., 1987; Bishop and Walker, 2003) thought to arise from strengthened * Corresponding author. Tel.: C1 313 577 5678; fax: C1 313 577 cellular transduction mechanisms within the direct 3125. striatal pathway (Cai et al., 2002; Gerfen et al., 2002; E-mail address: [email protected] (P.D. Walker). Papadeas et al., 2004). 5-HT2 receptor stimulation of

0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.03.008 C. Bishop et al. / Neuropharmacology 49 (2005) 350e358 351 the DA-depleted striatum with the 5-HT2 receptor 2. Methods agonist [(G)-1-(4-iodo-2,5-dimethoxyphenyl)-2-amino- propane] (DOI) also induces motor behaviors (Bishop 2.1. Animals and Walker, 2003; Bishop et al., 2004), likely as a consequence of increased 5-HT2A receptor expression Male SpragueeDawley rat pups (Charles River in the dorsal striatum (Radja et al., 1993; Laprade Laboratories, Wilmington, MA) were used and main- et al., 1996; Basura and Walker, 1999) and enhanced tained in strict accordance with the guidelines of the 5-HT2 receptor-mediated activation of the direct Institutional Animal Care and Use Committee of striatal pathway (Gresch and Walker, 1999a; Basura Wayne State University and the ‘‘Guide for the Care and Walker, 2000, 2001). and Use of Laboratory Animals’’ (Institute of Labora- Given that motor behavior likely recruits multiple tory Animal Resources, National Academy Press 1996; striatal neurotransmitter systems, recent attention has NIH publication number 85e23, revised 1996). Rat focused on the interaction between DA and 5-HT pups arrived on postnatal day 2 and were initially receptors. In the intact striatum, studies consistently group-housed (n Z 12/dam) in plastic cages (22 cm high, demonstrate that intrinsic 5-HT2 receptors can modify 45 cm deep and 23 cm wide) within a temperature DA function and have postulated divergent roles for controlled colony room (22e23 C) illuminated on 5-HT2A and 5-HT2C receptor subtypes (Lucas and a 12 h light/dark cycle (lights on 0700 hours). Once rats Spampinato, 2000; Porras et al., 2002). 5-HT2A were 3 weeks old, they were weaned from the dams, antagonists reduce hyperlocomotion induced by co- group-housed (4e5/cage) and fed standard lab chow caine, amphetamine and 3,4-methylenedioxymetham- (Rodent Diet 5001; LabDiet, Brentwood, MO) and phetamine (MDMA) (Kehne et al., 1996; O’Neill et al., water which were available throughout the remainder of 1999), conversely, 5-HT2C receptor antagonists have the experiment. been shown to enhance or reduce these effects depend- ing upon which compounds and neural sites are studied 2.2. 6-Hydroxydopamine (6-OHDA) lesions and (Filip and Cunningham, 2002; Fletcher et al., 2002; cannulae implantation surgeries Filip et al., 2004). While less is known about the interaction of these receptors following DA depletion, Bilateral intracerebroventricular (icv) 6-OHDA le- DA-related behaviors are also altered by 5-HT2 sions were performed as reported previously (Basura receptor antagonists. For instance, elevated oral and Walker, 1999). On postnatal day 3, rat pups were dyskinesia induced by ventral striatal infusion of the anesthetized to effect by hypothermia (body immersion partial D1 agonist SKF 38393 is lessened by 5-HT2A/2C in a foil chamber cooled in wet ice) and a midsagittal receptor antagonism (Gong et al., 1992; Plech et al., incision was made on the dorsal scalp to reveal the skull 1995). Recent work from our laboratory has also sutures and obtain reference coordinates from bregma demonstrated that hyperlocomotor activity induced by (Paxinos and Watson, 1998). Each rat received a bi- systemic or intrastriatal administration of the full D1 lateral infusion of 6-OHDA (60 mg; Sigma, St. Louis, agonist SKF 82958 to adult DA-depleted rats (O’Boyle MO) or its vehicle solution of saline (0.9% NaCl) et al., 1989) can be reduced by pretreatment with the containing 0.1% ascorbic acid (Sigma) at a rate of 5 ml 5-HT2A/2C receptor antagonist ritanserin (Bishop et al., over 30 s into each ventricle (AP 0.0 mm, ML G1.0 mm, 2003). However, to this point it is not known which DV ÿ2.5 mm relative to bregma) using a 10 ml syringe receptor subtype(s), 5-HT2A vs. 5-HT2C, contribute to equipped with a 30 gauge needle. Pups were placed this effect. under a heat lamp for recovery prior to being returned To determine this, the present study examined the to the mother rat. All rats received subcutaneous effects of 5-HT2A and 5-HT2C receptor antagonism on injections of desipramine (25 mg/kg; Sigma) 30 min hyperlocomotor activity elicited by striatal D1 receptor prior to surgery to protect norepinephrine (NE) stimulation in neonatal DA-depleted rats. Because neurons. Sixty days following icv lesions, rats were 5-HT2A receptor signaling is enhanced under conditions anesthetized with an intraperitoneal injection of ket- of DA depletion and thus are in a strengthened position amine (90 mg/kg; Lloyd Laboratories, Shenandoah, IO) to mediate motor activity, we predicted that antagonism and xylazine (15 mg/kg; Lloyd Laboratories) mixture with the putative 5-HT2A receptor antagonist M100907 and implanted bilaterally with chronic 22 gauge in- (Kehne et al., 1996), but not the purported 5-HT2C tracranial guide cannulae (C313G/SPC; Plastics One antagonist RS102221 (Bonhaus et al., 1997) would Inc., Roanoke, VA). With the incisor bar positioned decrease D1-induced hyperlocomotion. The results of 3.3 mm below the interaural line, cannulae were this study suggest that 5-HT2A but not 5-HT2C receptors positioned above the dorsal striatum bilaterally using within the DA-depleted striatum significantly reduce the coordinates AP C0.4 mm, ML G2.9 mm and DV the supersensitive locomotor responses to striatal D1 ÿ3.6 mm relative to bregma (Paxinos and Watson, agonism. 1998). Cannulae were fixed in place using liquid and 352 C. Bishop et al. / Neuropharmacology 49 (2005) 350e358 powder dental acrylic (Lang Dental, Wheeling, IL). At centimeters) and stereotypy activity (beam breaks the completion of surgery, guide cannulae were fitted counted at the same photocell array). Prior to experi- with 28 gauge inner stylets (Plastics One) to maintain mentation, rats were acclimated to the behavioral patency. procedure at least 3 times. All testing occurred between 0830 and 1230 hours. On testing days, rats were placed 2.3. Pharmacological treatments in the activity chambers for 30 min before injections commenced. During injections, rats were lightly re- The following D1 and 5-HT2 receptor agents were strained with a towel and 16 mm 30 gauge injectors used in this study: SKF 82958 ((G)6-chloro-7, (Plastics One) were slowly lowered to extend 2 mm past 8-dihydroxy-3-allyl-1-phenyl 2,3,4,5-tetra-hydro-(1 H)- the end of the guide cannulae. Once injectors were in 3-benzazepine hydrobromide; Sigma); M100907 place, drug was infused at a rate of 0.63 ml/min by ((R)-(C)-a-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl) a microinfusion pump (Harvard Apparatus, Boston, ethyl]-4-piperidinemethanol; synthesized as described MA) that held two-50 ml Hamilton syringes attached to in Ullrich and Rice, 2000); RS102221 (8-[5-(2,4- plastic tubing (PE20 Tygon tubing; Plastics One) and dimethoxy-5-(4-trifluoromethylsulpho-amido)phenyl-5- the injectors. Following infusions, injectors remained in oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione; Tocris, place for 30 s. Immediately after the injectors were Ellisville, MO). At least 1 week after cannulation, rats removed, rats were returned to the testing chambers for were randomly divided into treatment groups and behavioral recording, the pump was re-started and assigned to receive a regimen of bilateral intrastriatal injectors were inspected for fluid expulsion. drug infusions on separate testing occasions. One group of intact control (n Z 10) and DA-lesioned rats (n Z 10) 2.5. Tissue dissection and cryostat sectioning was tested for locomotor effects elicited by intrastriatal drug infusions of vehicle [50% dimethyl sulfoxide When experiments were completed, rats were sacri- (DMSO) in 0.9% NaCl] or SKF 82958 (0.1, 1.0 or ficed by decapitation and brains were immediately 10 mg). To determine whether these effects were more removed and dissected. Tissue from all rats included in likely to be mediated by 5-HT2A or 5-HT2C receptors, the study was examined for verification of striatal a larger group of control (n Z 24) and 6-OHDA- placements. To accomplish this, the region surrounding lesioned (n Z 24) rats was then tested for responses to the injection sites was blocked and rapidly frozen in vehicle or SKF 82958 (2.0 mg) alone or in combination isopentane (ÿ30 C) and stored at ÿ80 C. Cresyl violet with the 5-HT2C-preferring antagonist RS102221 (0.1 or (FD Neurotechnologies, Baltimore, MD) staining was 1.0 mg) or the 5-HT2A-preferring antagonist M100907 used to determine injection sites and neuronal viability (0.1 or 1.0 mg) in a counter-balanced design. Doses of from cryostat-generated 12 mm coronal sections sur- antagonists were based on previous work and they rounding the cannulae placements that were post-fixed showed pharmacological blockade of behaviors and no with 4% paraformaldehyde (Fisher Scientific, Hanover intrinsic effects on basal locomotor activity when Park, IL). To determine the level of DA depletion, administered alone (Filip and Cunningham, 2002; a randomly selected set of control (n Z 10) and 6- Bishop et al., 2004). All compounds were found to have OHDA-lesioned rat (n Z 10) striata were freshly dis- a pH in the range of 6e8 and were administered in sected C0.5 mm to ÿ0.5 mm around the injection site, a volume of 0.8 ml that preliminary dye tests demon- frozen at ÿ80 C and later subjected to monoamine strated was sufficient to limit the infusion to the striatal analysis using high performance liquid chromatography region of interest. The number of animals per group with electrochemical detection (HPLC-ED). ranged from 6 to 8 rats. No rat was tested more than 3 times, and at least 3 days separated each testing session. 2.6. HPLC analysis

2.4. Behavioral procedure HPLC-ED was performed on selected striatal tissue. To do so, samples were homogenized in a cold solution Locomotor activity monitoring was conducted in six containing 0.1 M perchloric acid, 1.0% ethanol, and identical acrylic chambers measuring 40 cm long, 40 cm 0.02% EDTA and centrifuged for 30 min at 4 C. wide and 30 cm high (Accuscan Instruments, Columbus, Supernatants from individual samples were analyzed OH). Each chamber was surrounded by a 15 ! 15 with reverse-phase HPLC-ED. The HPLC system infrared photocell array interfaced with a computer that consisted of a Waters WISP autoinjector, a Waters 510 ran the Versamax and Versadat programs (Accuscan dual piston pump, an external pulse dampener (Rainin), Instruments). These programs tabulated and processed a Waters guard pak column, and a C-18 (100 ! 3.2 mm, a number of variables related to locomotor behavior. 5 mm packing, Applied Biosystems) column. A mobile For the present experiment the variables collected over phase consisting of 0.75 mM sodium phosphate, twenty 3 min periods were total distance traveled (in 0.5 mM EDTA, 1.4 mM octane sulfonic acid, and 7% C. Bishop et al. / Neuropharmacology 49 (2005) 350e358 353 acetonitrile, pH 3.0 was used to separate out NE, DA, 5- HT and their metabolites. A coulometric detector (Model 5011, ESA) configured with three electrodes was used to measure the monoamines and their metabolites. An ESA model 5020 guard cell (C400 mV) was placed before the WISP injector and an ESA model 5011 analytical cell (first electrode at ÿ40 mV; second electrode at C500 mV) was placed immediately after the column. The signal from the second analytical electrode was recorded and analyzed by a Waters Baseline 810 Chromatography Workstation via a Waters Interface Module. The signals produced by the oxidation of monoamines and metabolites were measured and compared with those of known concentrations of high quality standards. The final oxidation current values were adjusted to milligram protein Fig. 1. Schematic representation of coronal sections of the rat brain amounts determined by Lowry protein assay and ex- depicting the distribution of striatal microinfusion sites. The section was taken from Paxinos and Watson (1998). Shaded and clear ovals pressed as nanogram monoamine per milligram protein. denote placements within the striatum for selected 6-OHDA lesion and intact control rats, respectively. Relevant anatomical structures are: Cc 2.7. Data analysis corpus callosum, Cpu caudate putamen, FL forelimb area of the cortex, LV lateral ventricle. All data collected were expressed as mean G S.E. Data collected from the SKF 82958 dose-response els in the 6-OHDA-injected group as compared to intact experiment were analyzed by 2-way analyses of variance control rats while DOPAC/DA ratio increased C421% (ANOVAs) with lesion and treatment as between indicating heightened DA turnover in a small percentage subjects factors. Data collected from 5-HT antagonist of surviving DA terminals. Increased 5-HT (C74%) and experiments were analyzed by 1-way ANOVAs with 5-hydroxyindole-3-acetic acid (5-HIAA) (C63%) levels treatment as a between subjects factor. When appropri- were indicative of the 5-HT hyperinnervation, but 5- ate, post hoc comparisons were carried out with Fisher’s HIAA/5-HT ratios remained at control levels. NE post hoc tests. For monoamine analysis, single group amounts in lesioned striata were not significantly (lesioned vs. intact) differences were determined by different from controls. Such results are consistent with independent 2-tailed t-tests. All analyses were carried our previous studies (Basura and Walker, 2001; Camp- out using Statistica software ’98 (Statsoft Inc., Tulsa, bell et al., 2001; Bishop et al., 2004). OK) with alpha values set to p ! 0.05.

3. Results 3.3. Locomotor eﬀects of intrastriatal SKF 82958

3.1. Representative histology Main effects of lesion (F(1,31) Z 9.88; p ! 0.05) and treatment (F(3,31) Z 5.24; p ! 0.05) on total distance Fig. 1 shows a schematic representation of a coronal demonstrated that 6-OHDA lesions increased ambula- brain section (Paxinos and Watson, 1998) located tion and that intrastriatal administration of the D1 0.48 mm anterior to bregma illustrating placements for agonist SKF 82958 produced significant effects on 20 randomly selected 6-OHDA-lesioned (shaded ovals) locomotor behavior (Fig. 2A). More importantly, post and control (clear ovals) rats included in the study. All hoc tests on a significant lesion ! treatment interaction rats analyzed were found to have injector placements (F(3,31) Z 3.19; p ! 0.05) indicated that both doses of within the dorsal aspects of the striatum. Upon SKF 82958 significantly enhanced ambulation in 6- histological examination, data from two rats (1 control; OHDA-lesioned rats compared to all other conditions 1 6-OHDA lesion) were removed due to exceptional (all p ! 0.05). On measures of stereotypy activity gliosis. (Fig. 2B) similar lesion (F(1,31) Z 13.22; p ! 0.05) and treatment main effects were found (F(3,31) Z 6.56; 3.2. Monoamine and metabolite levels p ! 0.05). Post hoc analyses of a significant lesion ! treatment interaction (F(3,31) Z 3.08; p ! 0.05) demon- As shown in Table 1, HPLC analyses revealed strated that the 10 mg dose of SKF 82958 increased a significant decline in striatal DA (ÿ99%) and stereotypy compared to all other groups (all p ! 0.05). 3,4-dihydroxyphenylacetic acid (DOPAC) (ÿ97%) lev- 6-OHDA-lesioned rats also displayed more stereotypy 354 C. Bishop et al. / Neuropharmacology 49 (2005) 350e358

Table 1 Effect of neonatal bilateral intracerebroventricular 6-hydroxydopamine (6-OHDA) treatment on concentrations of norepinephrine (NE), 3,4- dihydroxyphenylacetic acid (DOPAC), dopamine (DA), 5-hydroxyindolacetic acid (5-HIAA), serotonin (5-HT) and their metabolites/monoamine ratios in the striatum Group NE DOPAC DA DOPAC/DA 5-HIAA 5-HT 5-HIAA/5-HT Vehicle (n Z 10) 0.03 G 0.01 3.02 G 0.29 15.5 G 0.98 0.19 G 0.01 1.26 G 0.08 0.53 G 0.04 2.71 G 0.31 6-OHDA 0.04 G 0.01 0.08 G 0.01 0.12 G 0.02 0.99 G 0.16 2.05 G 0.14 0.92 G 0.08 2.38 G 0.16 (n Z 10) (133) (2.6)* (0.8)* (521)* (163)* (174)* (88) Values are nanogram monoamine or metabolite per milligram protein or ratios of metabolite to monoamine (mean G S.E.) with percent of vehicle group in parentheses. Differences between group means were determined by independent 2-tailed t-tests (*p ! 0.05 compared to vehicle group). following the 1.0 mg dose of SKF 82958 than either rats compared to all other conditions (all p ! 0.05). vehicle condition (both p ! 0.05). More importantly, both doses of M100907, when co- infused with SKF 82958 in 6-OHDA-lesioned rats, 3.4. Effects of intrastriatal 5-HT2A antagonism on reduced this effect (both p ! 0.05). As shown in Fig. 3B, D1-agonist induced locomotor behavior similar treatment effects were found on measures of stereotypy activity (F(7,53) Z 6.99; p ! 0.001). As was As shown in Fig. 3A, a significant main effect of observed with total distance, SKF 82958-induced treatment (F(7,53) Z 7.48; p ! 0.001) was found on total stereotypy in 6-OHDA rats ( p ! 0.05 vs. all other distance. Post hoc testing demonstrated that 2.0 mgof groups) was reduced by both doses of co-infused SKF 82958 induced locomotion in 6-OHDA-lesioned M100907 (both p ! 0.05).

A. Total distance 1500 A. Total distance Control 2000 Control Lesion * + Lesion 1000 1500 *

1000 500 + + Distance ± S.E.

Distance ± S.E. 500

0 0 0.1 1.0 10.0 0 Veh SKF SKF+ SKF+ SKF 82958 Dose ( µg) (2.0) M (0.1) M (1.0)

B. Stereotypy activity B. Stereotypy activity 1500 Control 2000 Control Lesion # Lesion 1500 1000 @ *

1000 + + 500 Counts ± S.E.

Counts ± S.E. 500

0 0 0 0.1 1.0 10.0 Veh SKF SKF+ SKF+ SKF 82958 Dose ( µg) (2.0) M (0.1) M (1.0)

Fig. 2. Locomotor effects of local infusion of the D1 agonist SKF Fig. 3. Locomotor effects of local co-infusion of the D1 agonist SKF 82958 (0, 0.1, 1.0 and 10 mg/side) into the striatum of intact control and 82958 (SKF; 2.0 mg/side) and the 5-HT2A antagonist M100907 (M; 0, 6-OHDA-lesioned rats. Graphs denote averages of twenty 3 min 0.1 and 1.0 mg/side) into the striatum of intact control and 6-OHDA- measurement periods for total distance G S.E. (A) and stereotypy lesioned rats. Graphs denote averages of twenty 3 min measurement activity G S.E. (B). Statistical differences were determined by 2-way periods of testing for total distance G S.E. (A) and stereotypy ANOVAs analyzing lesion and treatment effects. Significant post hoc activity G S.E. (B). Statistical differences were determined by 1-way testing differences are denoted by the following symbols: *p ! 0.05 vs. ANOVAs analyzing treatment effects. Significant post hoc differences all but Lesion-1.0, Cp ! 0.05 vs. all but Lesion-10.0, #p ! 0.05 vs. all, are denoted by the following symbols: *p ! 0.05 vs. Lesion-Veh and @p ! 0.05 vs. Control-0 and Lesion-0. Control-SKF, Cp ! 0.05 vs. Lesion-SKF. C. Bishop et al. / Neuropharmacology 49 (2005) 350e358 355

A. Total distance hyperlocomotor activity. More importantly, we extend 2000 what is currently known about D and 5-HT receptor Control * 1 2 Lesion interactions within the DA-depleted striatum by showing that D -induced locomotor activity can be 1500 * * 1 reduced by antagonism of striatal 5-HT2A, but not

1000 5-HT2C receptors. The striatum is a major component of the basal ganglia and plays an integral role in movement initiation

Distance ± S.E. 500 and execution. Under normal conditions, DA and 5-HT innervation from nigrostriatal and raphe-striatal aﬀer- 0 Veh SKF SKF+ SKF+ ents, respectively (Graybiel, 1990; Lavoie and Parent, (2.0) RS (0.1) RS (1.0) 1990) regulate its function. As such it has been postulated that striatal output is likely mediated by B. Stereotypy activity interactions between intrinsic DA and 5-HT receptors. 2000 Control For example, microdialysis studies have demonstrated Lesion that 5-HT2 receptor antagonists alter striatal extracel- 1500 lular DA levels (Lucas and Spampinato, 2000; Porras * * * et al., 2002), and locomotion induced by psychomotor 1000 stimulants (O’Neill et al., 1999; Fletcher et al., 2002; Filip et al., 2004).

Counts ± S.E. 500 Selective destruction of central DA through the administration of the catecholamine neurotoxin 6- 0 OHDA significantly modifies striatal DA and 5-HT Veh SKF SKF+ SKF+ (2.0) RS (0.1) RS (1.0) neurotransmission (Kostrzewa et al., 1998; Reader and Dewar, 1999). For example, D1 receptor agonist Fig. 4. Locomotor effects of local co-infusion of the D agonist SKF 1 treatment more potently stimulates ambulation and 82958 (SKF; 2.0 mg/side) and the 5-HT2C antagonist RS102221 (RS; 0, 0.1 and 1.0 mg/side) into the striatum of intact control and 6-OHDA- stereotypy (Breese et al., 1985, 1987; Bishop and lesioned rats. Graphs denote averages of twenty 3 min measurement Walker, 2003), postulated to occur as a result of G periods of testing for total distance S.E. (A) and stereotypy strengthened D1-related cellular signal transduction activity G S.E. (B). Statistical differences were determined by 1-way (Cai et al., 2002; Gerfen et al., 2002; Papadeas et al., ANOVAs analyzing treatment effects. Significant post hoc differences are denoted by the following symbol: *p ! 0.05 vs. Lesion-Veh and all 2004). 5-HT function is also altered when brain DA control treatments. levels are significantly reduced. For example, neonatal DA depletion induces 5-HT hyperinnervation (Breese et al., 1984; Stachowiak et al., 1984) and, as shown by 3.5. Effects of intrastriatal 5-HT2C antagonism on the present study and others, increased 5-HT levels in D1-agonist induced locomotor behavior striatum (Basura and Walker, 1999; Bishop et al., 2004). This enhanced 5-HT signaling, thought to contribute to Analysis of total distance (Fig. 4A) revealed a signif- hyperactivity in neonatal DA-depleted rats, can be icant main effect of treatment (F(7,52) Z 7.48; p ! 0.001). reduced by 5-HT2 receptor antagonism (Luthman As previously shown, post hocs showed that SKF 82958 et al., 1991). Interestingly, neither 5-HT2A nor 5-HT2C significantly enhanced locomotion in the 6-OHDA- receptor plasticity is dependent upon 5-HT hyperinner- ! lesioned rats ( p 0.05). However, in contrast to vation (Laprade et al., 1996; Basura and Walker, 1999; M100907, neither dose of RS102221, when co-infused Fox and Brotchie, 2000a), suggesting that enhanced 5- with SKF 82958 in 6-OHDA-lesioned rats, altered this HT levels are not required to elicit 5-HT2-specific effects. effect. As shown in Fig. 4B, post hoc testing of This is supported by studies showing that striatal a significant main effect of treatment for stereotypy infusion of the 5-HT receptor agonist DOI increases Z ! 2 activity (F(7,52) 8.80; p 0.001) revealed that SKF locomotor behaviors in DA-depleted rats (Bishop and ! 82958 increased stereotypy in 6-OHDA rats ( p 0.05) Walker, 2003; Bishop et al., 2004). These effects are that was unaffected by co-infused RS102221. thought to arise as a result of upregulated striatal 5- HT2A receptors (Radja et al., 1993; Laprade et al., 1996; Basura and Walker, 1999) and strengthened activation 4. Discussion of the direct striatal output pathway (Gresch and Walker, 1999a; Basura and Walker, 2000, 2001). In concert with previous studies, the results of the While it is clear that DA and 5-HT functions are present investigation demonstrated that striatal D1 ago- altered after DA depletion, less is known about how nist administration to 6-OHDA-lesioned rats induces these receptors interact under these conditions to 356 C. Bishop et al. / Neuropharmacology 49 (2005) 350e358 influence motoric behavior. In a series of papers, we have after intra-subthalamic administration of m-CPP in the demonstrated that striatal 5-HT2 receptors are in intact rat (Eberle-Wang et al., 1996). In the DA-depleted a strengthened position to influence supersensitive brain, the substantia nigra pars reticulata appears to be behavioral and cellular responses to D1 agonist admin- a particularly important site mediating 5-HT’s effects. istration. For example, gene expression studies have For example, nigral 5-HT2C receptor binding is elevated indicated that combined administration of the D1 agonist in the post-mortem tissue of Parkinson’s disease patients SKF 38393 and 5-HT2 receptor agonist DOI increases (Fox et al., 2000a). Functionally, rodent studies have activation of the direct striatonigral output pathway shown that intranigral administration of the 5-HT2C (Gresch and Walker, 1999b; Campbell et al., 2001). antagonist SB206553 induces contralateral rotations in These effects were recently corroborated with behavioral the unilateral DA-depleted rat (Fox et al., 1998). studies showing that intrastriatal co-administration of Moreover, systemic administration of SB206553 enhan- the full D1 agonist SKF 82958 and DOI, synergistically ces contralateral rotations elicited by D1 or D2 agonists increased locomotion (Bishop and Walker, 2003). (Fox et al., 1998; Fox and Brotchie, 2000b). Thus, it Further studies have also revealed that D1 agonist appears that extrastriatal 5-HT2C receptors may more supersensitivity following 6-OHDA lesions in adult rats readily impact D1 agonist-induced movement under can be reduced by prior administration of the 5-HT2A/2C conditions of DA depletion. antagonist ritanserin (Bishop et al., 2003). While these In the present study we employed the putative 5- studies suggest that 5-HT2 receptors can influence striatal HT2A and 5-HT2C receptor antagonists M100907 and D1 receptors, it was not known which receptor sub- RS102221 to discern the potential contributions of these typye(s) mediated these effects. receptors to D1 agonist-induced locomotor activity. A number of clues led us to hypothesize that striatal These compounds have nanomolar binding affinities for 5-HT2A receptors were more likely to influence D1- 5-HT2A vs. 5-HT2C receptors with 100-fold selectivity in related hyperlocomotor activity. First, in the intact discriminating each receptor subtype (Kehne et al., brain, 5-HT2A and 5-HT2C receptors have divergent 1996; Bonhaus et al., 1997). As demonstrated by the roles in modulating DA function. For example, 5-HT2A results of the present study, only M100907 was able to receptor antagonism decreases striatal DA release effectively reduce the D1-induced locomotor activity, (Lucas and Spampinato, 2000) and behavioral activity indicating that within the DA-depleted striatum, 5- accompanying psychomotor stimulants (O’Neill et al., HT2A receptors more likely influence the function of D1 1999; Fletcher et al., 2002). 5-HT2C receptor antagonism receptors. While it is not clear how these receptors enhances striatal DA levels (Lucas and Spampinato, interact, it is known that DA depletion increases the 2000), but can increase or decrease psychomotor coupling of D1 receptors to Gq/11 proteins which are stimulant-induced behavioral activity based on the site functionally linked to phosphoinositol hydrolysis and (accumbens vs. medial prefrontal cortex) and compound protein lipase C second messenger cascades (Cai et al., administered (RS102221 vs. SB242084) (Filip and 2002). Because 5-HT2A receptors utilize phosphoinositol Cunningham, 2002; Fletcher et al., 2002; Filip et al., hydrolysis as a second messenger (Berg et al., 1994), DA 2004). Second, DA depletion has been shown to increase depletion-induced alterations in D1 signal transduction levels of striatal 5-HT2A mRNA without altering 5- mechanisms may strengthen 5-HT2A/D1 cooperation. HT2C receptor mRNA (Laprade et al., 1996; Basura and While the present study demonstrated that striatal 5- Walker, 1999). Third, gene activation within the direct HT2A receptors functionally influence supersensitive D1 pathway of the DA-depleted rat stimulated by 5-HT receptors in the DA-depleted rodent, these findings may release agents or 5-HT agonism is blocked by 5-HT2A- have relevance to excessive movement in humans. For preferring antagonists (Basura and Walker, 2000, 2001). example, it has been demonstrated that 5-HT2A Finally, DOI-induced locomotor activity in neonate 6- receptors are highly expressed in the striosomes of the OHDA-lesioned rats was attenuated by the 5-HT2A and human caudate nucleus and putamen (Waeber and not 5-HT2C receptor antagonism (Bishop et al., 2004). Palacios, 1994; Lopez-Gimenez et al., 1999) and are Thus, the relationship between 5HT2A and D1 receptors postulated to play a significant role in the DA-mediated in the DA-depleted striatum appears strengthened. induction of stereotypy and unwanted movements While we did not find that striatal 5-HT2C receptor (Graybiel et al., 2000; Henry et al., 2003). Because antagonism altered D1-induced locomotor activity, overactivity of D1 receptors in the direct striatal output there is evidence that following DA depletion, enhanced pathway plays a central role in the expression of oral activity induced by the D1 agonist SKF 38393 and stereotypy (Starr and Starr, 1986; Chartoff et al., 2001) the 5-HT2A/2C agonist m-chlorophenylpiperazine (m- and the development of levodopa-induced dyskinesia CPP) is mediated through striatal 5-HT2C receptors (Andringa et al., 1999; Rascol et al., 2001), reducing (Gong and Kostrzewa, 1992; Plech et al., 1995). supersensitive D1-mediated signaling through a Extrastriatal 5-HT2C receptor effects on movement have non-dopaminergic mechanism like 5-HT2A receptor also been reported, including increased oral dyskinesia antagonism may allow for treatment of excessive move- C. Bishop et al. / Neuropharmacology 49 (2005) 350e358 357 ments without loss of therapeutic efficacy. For example, hyperlocomotion in 6-hydroxydopamine-lesioned rats. Neurosci- the atypical antipsychotic clozapine, which shows, ence 121, 649e657. among other receptor families, affinity as an antagonist Bishop, C., Kamdar, D.P., Walker, P.D., 2003. Intrastriatal serotonin 5-HT2 receptors mediate dopamine D1-induced hyperlocomotion at 5-HT2 receptor sites (Meltzer, 1999), and the in 6-hydroxydopamine-lesioned rats. Synapse 50, 164e170. 5-HT2A/2C receptor antagonist ritanserin, are reported Bishop, C., Tessmer, J.L., Ullrich, T., Rice, K.C., Walker, P.D., 2004. to reduce levodopa-induced dyskinesia in Parkinson’s Serotonin 5-HT2A receptors underlie increased motor behaviors disease patients (Maertens de Noordhout and Delwaide, induced in dopamine-depleted rats by intrastriatal 5-HT2A/2C 1986; Pierelli et al., 1998; Durif et al., 2004). agonism. Journal of Pharmacology and Experimental Therapeutics 310, 687e694. In summary, this study demonstrated that increased Bonhaus, D.W., Weinhardt, K.K., Taylor, M., DeSouza, A., locomotor behaviors induced by striatal administration McNeeley, P.M., Szczepanski, K., Fontana, D.J., Trinh, J., of the D1 agonist SKF 82958 in rodents with near total Rocha, C.L., Dawson, M.W., Flippin, L.A., Eglen, R.M., 1997. DA depletion can be reduced by co-infusion of the RS-102221: a novel high affinity and selective, 5-HT2C receptor e selective 5-HT antagonist M100907, but not the 5- antagonist. Neuropharmacology 36, 621 629. 2A Breese, G.R., Baumeister, A.A., McCown, T.J., Emerick, S.G., HT2C selective antagonist RS102221. Coupled with gene Frye, G.D., Crotty, K., Mueller, R.A., 1984. Behavioral differences expression studies, the results of this study suggest that between neonatal and adult 6-hydroxydopamine-treated rats to striatal D1 and 5-HT2A receptors interact to alter direct dopamine agonists: relevance to neurological symptoms in clinical striatal pathway output. This strengthened relationship, syndromes with reduced brain dopamine. Journal of Pharmacology e brought about by DA depletion may represent a novel and Experimental Therapeutics 231, 343 354. Breese, G.R., Baumeister, A., Napier, T.C., Frye, G.D., Mueller, R.A., mechanism by which pathological D1-related signaling 1985. Evidence that D-1 dopamine receptors contribute to the may be reduced and suggests an alternative pharmaco- supersensitive behavioral responses induced by L-dihydroxypheny- logical target for the lessening of excessive movements lalanine in rats treated neonatally with 6-hydroxydopamine. that can occur with DA replacement therapy. Journal of Pharmacology and Experimental Therapeutics 235, 287e295. Breese, G.R., Duncan, G.E., Napier, T.C., Bondy, S.C., Iorio, L.C., Mueller, R.A., 1987. 6-Hydroxydopamine treatments enhance behavioral responses to intracerebral microinjection of D1- and Acknowledgements D2-dopamine agonists into nucleus accumbens and striatum without changing dopamine antagonist binding. Journal of Special thanks to Drs. K.C. Rice and T. Ullrich for Pharmacology and Experimental Therapeutics 240, 167e176. the generous donation of the 5-HT antagonist Cai, G., Wang, H.Y., Friedman, E., 2002. Increased dopamine 2A receptor signaling and dopamine receptor-G protein coupling in M100907. This work was supported by the National denervated striatum. Journal of Pharmacology and Experimental Institute of Neurological Disorders & Stroke Grant Therapeutics 302, 1105e1112. NS39013 to P.D.W. Campbell, B.M., Gresch, P.J., Walker, P.D., 2001. 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