Role of Dopamine D4 Receptors in Motor Hyperactivity Induced by Neonatal 6-Hydroxydopamine Lesions in Rats Kehong Zhang, M.D., Ph.D., Frank I. Tarazi, Ph.D., and Ross J. Baldessarini, M.D. The role of dopamine D4 receptors in behavioral hyperactivity D4-selective antagonist CP-293,019 dose-dependently was investigated by assessing D4 receptor expression in brain reversed lesion-induced hyperactivity, and D4-agonist CP- regions and behavioral effects of D4 receptor-selective ligands 226,269 increased it. These results indicate a physiological in juvenile rats with neonatal 6-hydroxydopamine lesions, a role of dopamine D4 receptors in motor behavior, and may laboratory model for attention deficit-hyperactivity disorder suggest much-needed innovative treatments for ADHD. (ADHD). Autoradiographic analysis indicated that motor [Neuropsychopharmacology 25:624–632, 2001] hyperactivity in lesioned rats was closely correlated with © 2001 American College of Neuropsychopharmacology. increases in D4 but not D2 receptor levels in caudate-putamen. Published by Elsevier Science Inc. KEY WORDS: Attention deficit-hyperactivity disorder; Human D4 receptors occur in multiple forms with 2– Autoradiography; Dopamine; D4; 6-hydroxydopamine; 11 copies of a 16-amino acid (48 base-pair) sequence in Motor activity the putative third intracellular loop of the peptide (Van Dopamine (DA) modulates physiological processes Tol et al. 1992; Lichter et al. 1993; Asghari et al. 1994). through activation of five G-protein coupled receptors Several recent genetic studies indicate that the 7-repeat D4 receptor allele (D4.7), a relatively uncommon variant, of the D1-like (D1 and D5) and D2-like (D2, D3, and D4) re- ceptor families (Neve and Neve 1997). Since the cloning is more prevalent in patients with attention deficit- hyperactivity disorder (ADHD) (La Hoste et al. 1996; of their cDNA (Van Tol et al. 1991), D4 receptors have re- ceived much attention, in part because some atypical an- Bailey et al. 1997; Rowe et al. 1998; Faraone et al. 1999). Haplotype relative-risk analysis to assess allele trans- tipsychotics, notably clozapine, bind to D4 receptors with somewhat higher affinity than to the more preva- mission also suggests that the D4.7 receptor may be as- sociated with ADHD (Swanson et al. 1998). The same lent D2 receptors (Van Tol et al. 1991; Seeman et al. 1997; Tarazi et al. 1997; Tarazi and Baldessarini 1999). D4.7 phenotype is also linked to personality traits re- lated to ADHD, notably, novelty-seeking and impulsiv- ity (Benjamin et al. 1996; Ebstein et al. 1996; Comings et From the Mailman Research Center, McLean Division of Massa- al. 1999). Biochemical analysis of these D receptor vari- chusetts General Hospital, 115 Mill Street, Belmont, MA 02478, 4 USA; and Consolidated Department of Psychiatry and Neuro- ants indicates that D4.7 receptors are less efficient in science Program, Harvard Medical School, Boston, MA 02115 USA. transducing extracellular DA signals to intracellular Address correspondence to: Kehong Zhang, M.D., Ph.D., Mail- adenylyl cyclase than other forms with fewer repeat se- man Research Center, McLean Hospital, 115 Mill Street, Belmont, MA 02478, Tel.: 617-855-3222; Fax: 617-855-3479, E-mail: kz@mclean. quences (Asghari et al. 1995). harvard.edu ADHD is a neuropsychiatric syndrome frequently Received November 14, 2000; revised March 28, 2001; accepted found in school-aged children, especially boys, that is April 9, 2001. Online publication: 4/10/01 at www.acnp.org/citations/Npp characterized by excesses of hyperactive, inattentive, 041001102. and impulsive behavior (Barkley 1990). Salient features NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 5 © 2001 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/01/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(01)00262-7 NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 5 Dopamine D4 Receptors and Hyperactivity 625 of ADHD are commonly modeled in juvenile rats fol- hydrochloride was provided by Celgene (Warren, NJ). lowing neonatal lesioning with 6-hydroxydopamine (6- CP-226,269 and CP-293,019 were generously donated OHDA) which destroys DA projections to forebrain by Pfizer (Groton, CT). All other drugs and chemicals (Shaywitz et al. 1976). Such rats exhibit several charac- were purchased from Fisher Scientific (Dallas, TX) or teristics resembling core symptoms of ADHD, most no- Sigma Chemicals (St. Louis, MO). tably, motor hyperactivity that occurs selectively dur- ing the periadolescent period, gradually declines as Neonatal Lesioning lesioned rats mature, and seems to represent deficient adaptation to environmental stimuli (Erinoff et al. Sprague-Dawley rats (Charles River Labs., Wilming- 1979). The motor hyperactivity in this model can be ton, MA) were maintained under a 12-h artificial day- dose-dependently antagonized by psychostimulants light/dark schedule (on, 7 A.M.-7 P.M.), with free access that are commonly used to alleviate symptoms of to tap-water and standard rat chow. On postnatal day ADHD (Heffner and Seiden 1982; Luthman et al. 1989). (PD) 1, male pups were assigned to lactacting dam (10/ Hyperactivity and attentional deficits that occur in each). On PD 5, pups received a subcutaneous injection ADHD are also modeled with genetic knockout mice of desipramine hydrochloride (25 mg/kg). 45 Minutes that lack functional DA transporters (DAT) (Giros et al. later, pups randomly received an intracisternal injec- 1996; Gainedtinov et al. 1999). However, the patho- tion of vehicle (0.9% NaCl containing 0.1% ascorbic physiology of these models may be dissimilar, and may acid), or 6-OHDA hydrobromide (100 ␮g free base) un- or may not reflect mechanisms underlying clinical der hypothermal anesthesia (Shaywitz et al. 1976). Pups ADHD. Notably, the 6-OHDA lesioning model in- were returned to nursing dams immediately after the volves neonatal removal of DA with overgrowth of se- intracranial injections. A total of 210 rat pups were in- rotonin projections to forebrain (Kostrzewa et al. 1998), cluded in the study. All procedures were approved by whereas the DAT knockout mouse involves a loss of the the McLean Hospital Institutional Animal Care and Use major mechanism for inactivating DA (Gainedtinov et Committee, in compliance with applicable federal and al. 1999)—a mechanism that may be overexpressed in local guidelines for experimental use of animals. The clinical ADHD (Dougherty et al. 1999; Dresel et al. extent of lesioning was verified by quantifying DAT 2000). binding with [3H]␤-CIT (Kula et al. 1999) at the comple- Association of D4 receptor polymorphism with tion of behavioral experiments. ADHD led us to study the expression of D4 receptors in juvenile rats following neonatal 6-OHDA lesions using Behavioral Experiments quantitative autoradiography. DA D1-like and D2-like receptor binding was examined for comparison. Effects Motor activity was monitored individually for 90 of highly D4-selective agents on hyperactivity were min in the periadolescent period between PD 21 and 30, studied and compared to representative stimulants using an infrared photobeam activity monitoring sys- used to treat ADHD. tem (San Diego Instruments, San Diego, CA) connected with a microcomputer. Behavioral testings were con- ducted in a novel environment (17ϫ8ϫ8 inch transpar- MATERIALS AND METHODS ent plastic cages with 4ϫ8 horizontal infrared beams), usually between 10:00 and 16:00 h in the absence of Radioligands and Chemicals food and water, except for experiments involving noc- [3H]Nemonapride (R[ϩ]-7-chloro-8-hydroxy-3-methyl- turnal testing (at 22:00–04:00 h). Locomotor activity was 1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 85.5 Ci/ defined as breaking of consecutive photobeams, and ac- mmol) and [3H]SCH-23390 (R[ϩ]-2,3,4,5-tetrahydro- cumulated every 5 min. 3-methyl-5-phenyl-1H-3-benzazepin-7-ol; 81.4 Ci/ Test agents were dissolved in 0.9% saline or 35% mmol) were obtained from New England Nuclear 2-hydroxypropyl-␤-cyclodextrin, and given intraperito- (NEN; Boston, MA). [3H]␤-CIT ([–]-2-␤-carbometh- neally (i.p.) immediately prior to testing. Each subject oxy-3-␤-[4-iodophenyl]-tropane; 64.7Ci/mmol) was ob- was evaluated in two behavioral testing sessions (one tained from Tocris Cookson (Bristol, UK). Tritium-sen- with vehicle, the other with a test drug), separated by at sitive Hyperfilm and autoradiography standards were least three days, and in randomized order. Some rats from Amersham (Arlington Heights, IL). D-19 devel- given CP-293,019 were pretreated with methysergide oper and fixative were from Eastman-Kodak (Roches- (2 mg/kg, i.p.) 30 min before behavioral testing. ter, NY). (ϩ)-Amphetamine sulfate, 1,3-ditolylguani- dine (DTG), cis-flupenthixol dihydrochloride, 6-OHDA Receptor Autoradiography hydrobromide, ketanserin tartrate, S(–)-pindolol, and S(–)-sulpiride were from Sigma–Research Biochemicals Rats were sacrificed 48 h after the last behavioral test- International (RBI; Natick, MA). (ϩ)-Methylphenidate ing session (on PD 32) by rapid decapitation, and brains 626 K. Zhang et al. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 5 were quickly removed and frozen. Coronal brain sec- nocturnal testing (Figure 1, Panels A and B). Notably, tions (10 ␮m) were prepared in a cryostat at –17ЊC, thaw- motor activity of lesioned rats did not differ signifi- mounted on gelatin-coated microscopic slides and stored cantly from controls for the first 5–10 min of testing ses- at –80ЊC until quantitative autoradiographic assays. sion, but failed to decline throughout the 90 min ses- Densities of D2-like and D4 receptors were deter- sion, long after arousal in control rats had greatly mined autoradiographically as previously detailed and diminished. Hyperactivity in lesioned rats was reduced characterized pharmacologically (Tarazi et al. 1997, by both (ϩ)-amphetamine and (ϩ)-methylphenidate 1998a,b). Briefly, tissue sections were preincubated for (Figure 1, Panel C). In contrast to their motor-inhibiting 60 min at room temp in 50 mM Tris-HCl buffer contain- effects in lesioned rats, both psychostimulants greatly ing (mM): NaCl (120), KCl (5), CaCl2 (2), and MgCl2 (1).