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NEUROPHARMACOLOGY

Serotonin 5-HT_2A Receptors Underlie Increased Motor Behaviors Induced in Dopamine-Depleted Rats by Intrastriatal 5-HT2A/2C Agonism

Departments of Anatomy and Cell Biology (C.B., P.D.W.) and Psychiatry and Behavioral Neurosciences (J.L.T.), Wayne State University School of Medicine, Detroit, Michigan; and Laboratory of Medicinal Chemistry (T.U., K.C.R.), National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland

Received for publication January 30, 2004
Accepted March 24, 2004.

	Abstract

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Gene expression studies have suggested that dopamine (DA) depletion increases the sensitivity of striatal direct pathway neurons to the effects of serotonin (5-HT) via the 5-HT₂ receptor. The present study examined the possible influence(s) of 5-HT_2A or 5-HT_2C receptor-mediated signaling locally within the striatum on motor behavior triggered by 5-HT₂ receptor agonism in the neonatal DA-depleted rat. Male Sprague-Dawley rats were treated with 6-hydroxydopamine (6-OHDA; 60 µg in 5 µl per lateral ventricle) on postnatal day 3 to achieve near-total DA depletion bilaterally. Sixty days later, sham-operated (saline-injected) or 6-OHDA-treated rats were challenged with the 5-HT_2A/2C agonist DOI [(±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane] or saline either by systemic treatment or bilateral intrastriatal infusion. Motor behavior was quantified for 60 min after agonist injection using computerized activity monitors. Systemic DOI treatment (0.2 or 2.0 mg/kg i.p.) was more effective in inducing motor activity in the DA-depleted group compared with intact controls. Intrastriatal DOI infusion (1.0 or 10.0 µg/side) also produced a significant rise in motor activity in the DA-depleted group during the 30- to 60-min period of behavioral analysis but did not influence behavior in intact animals. The effects of intrastriatal DOI infusion were blocked by intrastriatal coinfusion of the 5-HT₂ antagonist ketanserin (1.0 µg) and the 5-HT_2A-preferring antagonist M100907 [(R)(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol; 1.0 µg] but not the 5-HT_2C-preferring antagonist RS102221 [8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione; 1.0 µg]. Such results support the hypothesis that 5-HT_2A receptor-mediated signaling events are strengthened within the striatum under conditions of DA depletion to provide a more potent regulation of motor activity.

Several studies indicate that 5-HT_2A receptors are positioned to mediate the influences of enhanced 5-HT signaling in the DA-depleted striatum. First, 5-HT release agents and 5-HT₂ receptor agonists gain potency in inducing striatal preprotachykinin (PPT; encodes substance P and neurokinin A gene expression within the direct striatal output pathway) under conditions of DA depletion (Gresch and Walker, 1999; Basura and Walker, 2000, 2001). Second, 5-HT₂ receptor-mediated signaling appears to contribute to spontaneous hyperactivity observed in neonatal DA-depleted rats (Luthman et al., 1991) and also fosters D₁ receptor-mediated hyperlocomotive behaviors following adult DA lesions (Bishop et al., 2003; Bishop and Walker, 2003). Third, similar to the 5-HT sprouting phenomenon, the 5-HT_2A receptor is more heavily expressed within the dorsal striatum following neonatal DA depletion (Radja et al., 1993; Laprade et al., 1996; Basura and Walker, 2001). These convergent results have led us to hypothesize that 5-HT_2A receptor stimulation may underlie hyperlocomotive behaviors in the DA-depleted rat.

The present study examined the effects of 5-HT₂ receptor agonism on motor behaviors expressed in adult rats that had received bilateral DA lesions as neonates. Although 5-HT₂ receptor agonists such as DOI [(±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane] produce minimal effects on locomotor behavior in the intact rat (Koskinen et al., 2000), we predicted that systemic DOI treatments would increase locomotor behaviors in 6-OHDA-treated rats since 5-HT₂ receptor stimulation has previously been shown to gain potency in regulating direct pathway gene expression (Gresch and Walker, 1999; Basura and Walker, 2001). To limit the effects of DOI to the striatal region that demonstrates prominent 5-HT_2A receptor plasticity, we measured behavior in response to local DOI infusion into the dorsal striatum of intact and DA-depleted groups. These latter experiments also included the coinfusion of selective antagonists M100907 (Johnson et al., 1996; Kehne et al., 1996) and RS102221 (Bonhaus et al., 1997) to discern the effects of local striatal 5-HT_2A versus 5-HT_2C receptor activation, respectively, on DOI-induced motor activity. The results obtained suggest that 5-HT_2A receptor plasticity within the dorsal striatum is functionally significant and may influence motor control mechanisms of the DA-depleted rodent.

	Materials and Methods

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Animals. Male Sprague-Dawley rat pups (Charles River Laboratories, Inc., Wilmington, MA) were used and maintained in strict accordance with the guidelines of the Institutional Animal Care and Use Committee of Wayne State University and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996). Rat pups arrived on postnatal day 2 and were initially group-housed (n = 12/dam) in plastic cages (22 cm high, 45 cm deep, and 23 cm wide) within a temperature-controlled colony room (22-23°C) illuminated on a 12-h light/dark cycle (lights on 7:00 AM). Once rats were 3 weeks old, they were weaned from the dams, group-housed (4-5/cage), and fed standard lab chow (Rodent Diet 5001; LabDiet, Brentwood, MO) and water, which were available throughout the remainder of the experiment.

6-OHDA Lesions and Cannulae Implantation Surgeries. Bilateral intracerebroventricular 6-OHDA lesions were performed as reported previously (Basura and Walker, 1999). On postnatal day 3, rat pups were anesthetized to effect by hypothermia (body immersion in a foil chamber cooled in wet ice), and a midsagittal incision was made on the dorsal scalp to reveal the skull sutures and obtain reference coordinates from bregma. Each rat received a bilateral infusion of 6-OHDA (60 µg; Sigma-Aldrich, St. Louis, MO) or its vehicle solution of saline containing 0.1% ascorbic acid (Sigma-Aldrich) at a rate of 5 µl over 30 s into each ventricle (anterior/posterior, 0.0 mm; medial/lateral, ±1.0 mm; dorsal/ventral, -2.5 mm relative to bregma) using a 10-µl syringe equipped with a 30-gauge needle. Pups were placed under a heat lamp for recovery prior to being returned to the mother rat. All rats received subcutaneous injections of desipramine (25 mg/kg; Sigma-Aldrich) 30 min prior to surgery to protect norepinephrine neurons. Sixty days following i.c.v. lesions, rats were anesthetized with an i.p. injection of ketamine (90 mg/kg; Lloyd Laboratories, Shenandoah, IA) and xylazine (15 mg/kg; Lloyd Laboratories) mixture and implanted bilaterally with chronic 22-gauge intracranial guide cannulae (C313G/SPC; Plastics One Inc., Roanoke, VA). With the incisor bar positioned 3.3 mm below the interaural line, cannulae were positioned above the dorsal striatum bilaterally using the coordinates anterior/posterior, +0.4 mm; medial/lateral, ±2.9 mm; and dorsal/ventral, -3.6 mm relative to bregma (Paxinos and Watson, 1998). Cannulae were fixed in place using liquid and powder dental acrylic (Lang Dental, Wheeling, IL). At the completion of surgery, guide cannulae were fitted with 28 gauge inner stylets (Plastics One) to maintain patency.

Pharmacological Treatments. The following 5-HT₂ receptor agents were used in this study: DOI (Sigma-Aldrich), ketanserin (Sigma-Aldrich), M100907 (synthesized as described by Ullrich and Rice, 2000), and RS102221 (Tocris Cookson Inc., Ellisville, MO). At least 1 week after cannulation, rats were randomly divided into treatment groups and assigned to receive a regimen of systemic and/or bilateral intrastriatal drug infusions on separate testing occasions. Intact control and DA-lesioned rats (n = 20) received an i.p. injection of saline vehicle or the 5-HT_2A/2C agonist DOI using 0.2 or 2.0 mg/kg doses in a repeated-measures design with groups counter-balanced to avoid order effects. For local manipulations, rats (n = 50) were tested for locomotor effects elicited by intrastriatal drug infusions of saline or DOI (1.0 or 10 µg) at a volume of 0.8 µl/side. To determine whether the effects of DOI were more likely to be mediated by 5-HT_2A or 5-HT_2C receptors, these rats were then tested for responses to vehicle (10% dimethyl sulfoxide in saline), 1.0 µg of DOI, 1.0 µg of the 5-HT₂ antagonist ketanserin, 1.0 µg of the 5-HT_2C-preferring antagonist RS102221, 1.0 µg of the 5-HT_2A-preferring antagonist M100907, or DOI coinfused with each of these antagonists (1.0 µg of agonist + 1.0 µg of antagonist) at a volume of 0.8 µl/side in a counterbalanced design. The number of animals per group ranged from 7 to 10 rats. No rat was tested more than three times, and at least 3 days separated each testing session.

Behavioral Procedure. Locomotor activity monitoring was conducted in six identical acrylic chambers measuring 40 cm long, 40 cm wide, and 30 cm high (AccuScan Instruments, Inc., Columbus, OH). Each chamber was surrounded by a 15 x 15 infrared photocell array interfaced with a computer that ran the Versamax and Versadat programs (AccuScan Instruments, Inc.). These programs tabulated and processed a number of variables related to locomotor behavior. For the present experiment, the variables collected over twenty 3-min periods were horizontal activity (beam breaks counted across the photocell array), distance traveled (in centimeters), movement bouts (number of times a rat ended and then initiated another movement period), stereotypy activity (beam breaks counted at the same photocell array), and stereotypy bouts (number of times a rat ended and then initiated another stereotypy period). Prior to experimentation, rats were acclimated to the behavioral procedure at least three times. All testing occurred between 8:30 AM and 12:30 PM. On testing days, rats were placed in the activity chambers for 30 min before injections commenced. During injections, rats were lightly restrained with a towel, and 15-mm 30-gauge injectors (Plastics One) were slowly lowered to extend 1 mm past the end of the guide cannulae. Once injectors were in place, drug was infused at a rate of 0.63 µl/min by a microinfusion pump (Harvard Apparatus Inc., Holliston, MA) that held two 50-µl Hamilton syringes attached to plastic tubing (PE20 Tygon tubing; Plastics One) and the injectors. Following infusions, injectors remained in place for 30 s. Immediately after the injectors were removed, the pump was restarted, and injectors were inspected for fluid expulsion.

Tissue Dissection and Cryostat Sectioning. When experiments were completed, rats were sacrificed by decapitation, and brains were immediately removed and dissected. Tissue from 20 randomly selected rats (10 control and 10 6-OHDA lesions) was used solely for histological examination. To accomplish this, the region surrounding the injection sites was blocked and rapidly frozen in isopentane (-30°C) and stored at -80°C. Cresyl violet (FD Neurotechnologies, Baltimore, MD) staining was used to determine injection sites and neuronal viability from cryostat-generated 12-µm coronal sections surrounding the cannulae placements that were postfixed with 4% paraformaldehyde (Fisher Scientific Co., Pittsburgh, PA). In another randomly selected set of control (n = 13) and 6-OHDA-lesioned rats (n = 13), striata were freshly dissected +0.5 mm to -0.5 mm around the injection site, frozen at -80°C, and later subjected to monoamine analysis using high-performance liquid chromatography (HPLC) with electrochemical detection to determine levels of DA depletion.

HPLC Analysis. HPLC with electrochemical detection was performed on striatal tissue as previously described (Gresch and Walker, 1999). 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 milligrams of protein amounts determined by Lowry protein assay and expressed as nanograms of monoamine per milligram of protein (mean ± S.E.).

Data Analysis. All behavioral data were expressed as means ± S.E. and analyzed by one-way analysis of variance (ANOVA) using Statistica software '98 (StatSoft, Tulsa, OK). When appropriate, post hoc comparisons were carried out with Fisher's least square design post hoc tests. For monoamine analysis, single-group (lesioned versus intact) differences were determined by independent two-tailed Student's t tests. Alpha values were set to p < 0.05.

	Results

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Representative Histology. Fig. 1 shows a schematic representation of a coronal brain section (Paxinos and Watson, 1998) located 0.48 mm anterior to bregma illustrating placements for control (clear ovals) and 6-OHDA-lesioned (shaded ovals) rats included in the study. All rats analyzed were found to have injector placements within the dorsal aspects of the striatum.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Schematic representation of coronal sections of the rat brain depicting the distribution of striatal microinfusion sites. The section was taken from Paxinos and Watson (1998). Shaded and clear ovals denote placements within the striatum for selected 6-OHDA-lesioned and intact control rats, respectively. Relevant anatomical structures are: Cc, corpus callosum; Cpu, caudate putamen; FL, forelimb area of the cortex; and LV, lateral ventricle.

Monoamine and Metabolite Levels. As shown in Table 1, HPLC analyses revealed a significant decline in striatal DA (-98%) and DOPAC (-94%) levels in the 6-OHDA-injected group compared with intact control rats, whereas the DOPAC/DA ratio increased (+213%), indicating heightened DA turnover in a small percentage of surviving DA terminals. Increased 5-HT (+95%) and 5-HIAA (+82%) levels were indicative of the 5-HT hyperinnervation phenomenon, but 5-HIAA/5-HT ratios remained at control levels. Norepinephrine amounts in lesioned striata were not significantly different from controls. Such results are consistent with our previous studies (Basura and Walker, 2000, 2001; Campbell et al., 2001).

Locomotor Effects of Systemic DOI. Peripheral administration of 5-HT₂ receptor agonists has been reported to have subtle but significant effects on motor activity (Willins and Meltzer, 1997). To this point, however, it is not known whether DA depletion modifies these effects. In the present study, systemic administration of DOI produced a number of dose-dependent effects on locomotor behavior that were enhanced by 6-OHDA lesions (Fig. 2), including horizontal activity [F_(5,57) = 8.91; p < 0.001], total distance [F_(5,57) = 8.33; p < 0.001], movement bouts [F_(5,57) = 11.35; p < 0.001], stereotypy activity [F_(5,57) = 6.43; p < 0.001], and stereotypy bouts [F_(5,57) = 11.01; p < 0.001]. Post hoc tests on each variable demonstrated that 6-OHDA-lesioned rats receiving either 0.2 or 2.0 mg/kg DOI were more active than if they received vehicle (all p < 0.05). In contrast, control rats only displayed enhanced activity following 2.0 mg/kg DOI on the variables movement bouts and stereotypy bouts (both p < 0.05). Furthermore, 6-OHDA-lesioned rats were found to be more active than controls on all variables following 2.0 mg/kg DOI (all p < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Motor effects of systemic DOI (0, 0.2, and 2.0 mg/kg i.p.) in intact control and 6-OHDA-lesioned rats. Graphs denote averages of twenty 3-min measurement periods for horizontal activity ± S.E. (A), total distance ± S.E. (B), movement bouts ± S.E. (C), stereotypy activity ± S.E. (D), and stereotypy bouts ± S.E. (E). Statistical differences were determined by one-way ANOVAs analyzing treatment effects. Post hoc comparisons on a limited number of variables are denoted by the following symbols: *, p < 0.05 versus vehicle-lesion; +, p < 0.05 versus vehicle-control; , p < 0.05 versus 0.2-lesion and 2.0-control.

Effects of Intrastriatal 5-HT₂ Agonism on Locomotor Behavior. To determine whether the effects of systemic DOI treatment may have been mediated by neurons of the striatum, we infused DOI into the dorsal striatum of control and 6-OHDA-lesioned rats (Fig. 3). Initial analyses across the entire 60-min postinfusion period yielded no treatment effects (data not shown); however, it was noted that during the second 30 min of measurement, DOI-treated rats appeared more active. As such, data reported here represent means for the second 30 min of treatment. ANOVAs on treatment effects revealed significant effects on horizontal activity [F_(5,49) = 2.68; p < 0.05], total distance [F_(5,49) = 2.80; p < 0.05], stereotypy activity [F_(5,49) = 2.68; p < 0.05], and stereotypy bouts [F_(5,49) = 3.16; p < 0.05] but not movement bouts. Post hoc testing demonstrated that 6-OHDA rats infused with either 1.0 or 10 µg DOI were more active than if they were infused with vehicle (all p < 0.05). Importantly, 6-OHDA-lesioned rats were more active than controls following 1.0 (all p < 0.05) or 10 (all p < 0.05) µg of DOI, with the exception of stereotypy activity, where no differences were found with the 10-µg dose of DOI.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Motor effects of local infusion of DOI (0, 1.0, and 10 µg/side) into dorsal striatum of intact control and 6-OHDA-lesioned rats. Graphs denote averages of ten 3-min measurement periods in the second 30 min of testing for horizontal activity ± S.E. (A), total distance ± S.E. (B), movement bouts ± S.E. (C), stereotypy activity ± S.E. (D), and stereotypy bouts ± S.E. (E). Statistical differences were determined by one-way ANOVAs analyzing treatment effects. Post hoc comparisons on a limited number of variables are denoted by the following symbols: *, p < 0.05 versus vehicle-lesion; +, p < 0.05 versus 1.0-control; and , p < 0.05 versus 10-control.

Effects of 5-HT₂ Receptor Antagonists on DOI-Stimulated Behavior. Because some of the locomotor effects of DOI originated from the striatum, the receptor specificity of these effects were explored. The lower dose (1.0 µg) of DOI was coinfused into the striatum with ketanserin (greater selectivity for 5-HT_2A versus 5-HT_2C), RS102221 (greater selectivity for 5-HT_2C versus 5-HT_2A), or M100907 (greater selectivity for 5-HT_2A versus 5-HT_2C) of intact and 6-OHDA-lesioned rats (Fig. 4). The effects of each antagonist alone were also measured. Data reported here also represent means for the second 30-min period of treatment. ANOVAs revealed significant treatment effects on horizontal activity [F_(15,125) = 3.29; p < 0.001], total distance [F_(5,125) = 1.76; p < 0.05], movement bouts [F_(15,125) = 2.54; p < 0.01], and stereotypy activity [F_(15,125) = 1.78; p < 0.05] but not stereotypy bouts. Although motor activity tended to be lower in lesioned rats treated with vehicle versus intact controls, these differences were not statistically significant. Post hoc testing reinforced our previous findings by showing that 6-OHDA rats infused with 1.0 µg of DOI were more active than if they were infused with vehicle or compared with control rats receiving 1.0 µg of DOI (all p < 0.05). Although alone no antagonist influenced activity in either lesion or control groups, ketanserin and M100907 significantly attenuated DOI-induced activity on each variable (all p < 0.05). In contrast, RS102221 did not reduce DOI-induced activity in 6-OHDA-lesioned rats on measures of total distance, movement bouts, or stereotypy activity.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Motor effects of vehicle (Veh), DOI (D; 1.0 µg), ketanserin (K; 1.0 µg), RS102221 (R; 1.0 µg), M100907 (M; 1.0 µg), or DOI coinfused with each antagonist (K+D, R+D, and M+D; 1.0 µg + 1.0 µg) into the dorsal striatum of intact control and 6-OHDA-lesioned rats. Graphs denote averages of ten 3-min measurement periods in the second 30 min of testing for horizontal activity ± S.E. (A), total distance ± S.E. (B), movement bouts ± S.E. (C), stereotypy activity ± S.E. (D), and stereotypy bouts ± S.E. (E). Statistical differences were determined by one-way ANOVAs analyzing dose effects. Post hoc comparisons on a limited number of variables are denoted by the following symbols: *, p < 0.05 versus all; +, p < 0.05 versus all Lesion conditions except R + D; , p < 0.05 versus vehicle-lesion but not DOI-lesion.

	Discussion

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The present study showed that systemic 5-HT₂ agonist treatment more potently stimulates motor activity within DA-depleted rats compared with intact controls. When DOI was confined to the striatum via local infusion, hyperactivity was again observed in DA-depleted rats but not in intact controls. The locomotor effects of intrastriatal DOI were completely suppressed by coinfusion of the nonselective 5-HT₂ receptor antagonist ketanserin as well as the selective 5-HT_2A receptor antagonist M100907 but not by the purported 5-HT_2C antagonist RS102221. These results suggest that striatal 5-HT_2A receptors underlie increased motor behaviors in the DA-depleted rat induced by local striatal 5-HT₂ receptor stimulation. Coupled with prior gene expression analysis, this study implicates the direct striatal output pathway as the circuit that underlies the heightened locomotor behavior.

In the intact rat, a well characterized motoric behavior induced by systemic DOI treatment is the head-twitch response (Darmani et al., 1992), now known to be mediated by 5-HT_2A receptors of the medial prefrontal cortex (Willins and Meltzer, 1997). In contrast, systemic DOI alone seems to produce only modest effects on locomotor behavior (Koskinen et al., 2000). Such results are consistent with observations of the present study showing that only movement bouts and stereotypic activity were increased in intact animals treated with the higher systemic DOI dose (2.0 mg/kg). 5-HT_2A and 5-HT_2C receptor-mediated signaling mechanisms within the basal ganglia have been suggested to provide significant regulatory control over motor activity when combined with other compounds such as DA stimulants (O'Neill et al., 1999; McMahon et al., 2001; Filip and Cunningham, 2002). In addition, previous studies have implicated the 5-HT₂ receptor system in spontaneous hyperactivity induced by neonatal DA depletion (Luthman et al., 1991). This latter observation is in agreement with the findings of the present study that lower systemic DOI doses were potent inducers of ambulation and stereotypic movements in DA-depleted animals.

It is interesting that neither low nor high doses of DOI induced a behavioral change in the intact animal following intrastriatal infusion in the present study. This result is not surprising given the fact that the rostral striatum exhibits weak DOI binding under normal conditions (McKenna et al., 1989; Appel et al., 1990). In agreement, direct pathway expression of PPT mRNA was unaffected in the rostral striatum following systemic DOI treatment but was increased in the caudal striatum where 5-HT_2A receptors are more heavily expressed (Walker et al., 1996; Gresch and Walker, 1999; Basura and Walker, 2001). Thus, although DOI may not be a strong regulator of locomotor behavior controlled by direct pathway neurons of the intact rostral striatum, it may have a greater influence if placed into the caudal striatum where PPT mRNA is more readily induced by this agonist. In contrast to the results of intrastriatal DOI infusions in the intact animal, the present study found that both low and high DOI doses induced locomotor and stereotypic behavior when infused into the rostral striatum of DA-depleted rats. This result is consistent with our previous observations that DOI provided a more effective regulation of striatal PPT mRNA expression in DA-depleted animals (Gresch and Walker, 1999; Basura and Walker, 2001). Increased 5-HT_2A receptor biosynthesis in the DA-depleted rostral striatum, as indicated by an increased level of [¹²⁵I]DOI binding and 5-HT_2A receptor mRNA expression (Radja et al., 1993; Laprade et al., 1996; Basura and Walker, 1999), may provide the means for the enhanced actions of DOI within the DA-depleted striatum. Together, these results strengthen our hypothesis that motor behavior induced in DA-depleted rats following local striatal 5-HT₂ receptor stimulation involves the activation of the direct striatal output pathway.

Antagonists were included in the design of this study that have nanomolar binding affinities for 5-HT_2A versus 5-HT_2C receptors with 100-fold selectivity in discriminating each subtype (Johnson et al., 1996; Kehne et al., 1996; Bonhaus et al., 1997). Although the present study did not determine whether antagonist concentrations within the injection site were in the range that would ensure selectivity, results showing the differential blocking effects of each antagonist help discern which receptor is likely involved in DOI-induced motor activities. Such data are important because although (±)-DOI binds selectively to 5-HT₂ receptors, this agonist is unable to discriminate between 5-HT_2A and 5-HT_2C subtypes (McKenna et al., 1989; Appel et al., 1990). Though 5-HT_2C receptors do not appear to be increased within the striatum in response to DA depletion (Basura and Walker, 1999), they are thought to provide significant regulation of signal transduction when stimulated (Wolf and Schutz, 1997). Interestingly, 5-HT_2C antagonists administered locally into the substantia nigra have been shown to potentiate movements induced by DA receptor agonists in the DA-depleted rat (Fox et al., 1998). However, intrastriatal infusion of the selective 5-HT_2C antagonist RS102221 (Bonhaus et al., 1997) had no effect on motor activity in the present study, whereas ketanserin and M100907 both suppressed DOI-induced movements in DA-depleted rats. Ketanserin was originally developed as a selective 5-HT₂ receptor antagonist, but it is also known to possess significant histamine and 1-adrenergic binding properties (Leysen et al., 1982). However, ketanserin is still useful to help discriminate between 5-HT₂ subtypes since it demonstrates superior antagonist properties at 5-HT_2A versus 5-HT_2C receptors (Jerman et al., 2001). The more recent development of M100907 (formerly MDL 100,907) has aided in further delineation of 5-HT₂ receptor subtypes since it binds the 5-HT_2A receptor with subnanomolar affinity in contrast to moderate to low (>100 fold separation) affinity at the 5-HT_2C and ₁-adrenergic sites and >500-fold separation from D₂ receptor binding (Johnson et al., 1996; Kehne et al., 1996). Thus, our observations that two 5-HT_2A receptor antagonists showed blocking effects whereas a 5-HT_2C receptor antagonist was without effect supports our hypothesis that a strengthened 5-HT_2A receptor system underlies the ability of intrastriatal DOI to produce movement in the DA-depleted rat. In accordance with these results, previous studies demonstrated that ketanserin suppressed the effects of systemic DOI treatment on striatal PPT mRNA induction in the DA-depleted rat (Basura and Walker, 2001).

Finally, although the present findings were uncovered in rodents, the results obtained may have relevance to human disorders of movement. Since the triggering mechanism for striatal 5-HT_2A receptor plasticity in rodents is DA depletion and not 5-HT sprouting (Laprade et al., 1996; Basura and Walker, 1999), enhanced 5-HT_2A receptor signaling may influence striatal function in adult disorders that deplete DA but do not necessarily induce the 5-HT hyperinnervation phenomenon. It may be significant that 5-HT_2A receptors are intensely expressed within striosomes of the human caudate nucleus and putamen (Waeber and Palacios, 1994; Lopez-Gimenez et al., 1999) and thus may play a prominent role in the regulation of striatal function. Such approximation may be relevant when considering the possibility that oversensitive 5-HT_2A receptors may contribute to unwanted movements, such as dyskinesias that develop in response to levodopa treatment in Parkinson's disease or excessive behaviors that accompany attention-deficit hyperactivity disorder. Indeed, recent evidence obtained from DA-depleted rodents suggests that striatal 5-HT_2A receptor plasticity contributes to abnormal movements in response to intrastriatal D₁ receptor stimulation (Bishop and Walker, 2003; Bishop et al., 2003). Therefore, although the phenomenon of 5-HT_2A receptor plasticity has been known for some years in rodents, the most interesting findings relevant to human movement disorders are yet to be revealed.

In summary, this study demonstrated that hyperlocomotive behaviors in rodents with near-complete depletion of DA were elicited by systemic or local intrastriatal infusion of the 5-HT2 receptor agonist DOI. The behavioral effects of intrastriatal DOI infusion in DA-depleted rats were completely suppressed by coinfusion of the 5-HT_2A receptor antagonists M100907 or ketanserin but not the 5-HT_2C receptor antagonist RS102221. Since the direct striatal output pathway has been shown to be sensitive to 5-HT_2A receptor stimulation under conditions of DA depletion, future studies will examine the involvement of this pathway in hyperkinetic behaviors that may be relevant to human movement disorders currently treated with DA stimulants.

	Acknowledgements

 
The authors thank Gregory Daut for technical assistance on this project.

	Footnotes

 
This work was supported by Grant NS39013 from the National Institute of Neurological Disorders and Stroke to P.D.W.

DOI: 10.1124/jpet.104.066365.

ABBREVIATIONS: DA, dopamine; 5-HT, 5-hydroxytryptamine (serotonin); PPT, preprotachykinin; DOI, (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane; 6-OHDA, 6-hydroxydopamine; M100907, (R)-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol; RS102221, 8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione; ANOVA, analysis of variance; DOPAC, 3,4-dihydroxyphenlyacetic acid; 5-HIAA, 5-hydroxyindole-3-acetic acid.

¹ Current Address: Novartis Research Institute, A-1235 Vienna, Austria.

Address correspondence to: Dr. Paul D. Walker, Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. E-mail: pdwalker{at}med.wayne.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

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