Short Communication Altering behavioral responses and dopamine transporter protei antisense peptide nucleic acids Beth M. Tyler-McMahon a, *, Jennifer A. Stewart a , Joshua Jackson a , M.D.Bitner b , Abdul Fauq b , Daniel J. McCormick c , Elliott Richelson a a Neuropsychopharmacology, Mayo Clinic, Birdsall Medical Research Building, 4500 San Pablo Rd., Jacksonville, FL 32224, USA b Neurochemistry, Mayo Clinic, 4500 San Pablo Rd., Jacksonville, FL 32224, USA c Biochemistry and Molecular Biology, Mayo Clinic, 200 First St., SW,Rochester, MN 55905, USA Received 29 June 2000; accepted 19 December 2000 Abstract The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scramb or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged wi amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant incr motility (P , 0.01). When these rats were challenged with amphetamine, motility ofthe saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals sho only 3.4-fold (P , 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared wi scrambled PNA, and mismatch PNA controls (P , 0.001). These results extend our previous findings that brain proteins ca down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some no for the treatment of diseases dependent upon the function of the dopamine transporter. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Peptide nucleic acids; Dopamine transporter; Locomotor behavior; Antisense 1.Introduction The DAT, a member of the Na/Cl-dependent transporters containing 12 transmembrane domains [1], is thoughto control both the spatial and temporal activity of released dopamine by rapidly transporting dopamine into presynap- tic terminals [2]. The dopamine system has been implicated in the control of locomotion, cognition, and neuroendocrine functions [3]. The DAT is a target of psychoactive drugs as well as drugsof abuse,including cocaine and amphet- amines. Otherinvestigators have used knockout mice,in which the DAT gene was inactivated, to establish the crit- ical role of DAT in controlling dopamine levels and loco- motion, and its role as an obligatory target for cocaine and amphetamines [2,3]. These knockout mice,in which both copies of the DAT were inactivated, exhibited spontaneous hyperlocomotion and indifference to the behavioral effects of both cocaineand amphetamines. Interestingly, these knockouts still self-administered cocaine [4]. We were interested to see if we could knock down the expression of DAT using antisense methods. Previously, w showed that peptide nucleic acids can be used to knock down expression of receptors in the brain [5,6]. Others ha also reported in vivo success using PNAs directed at d -opi- oid [7] and galanin receptors [8]. In addition, despite prob lems with poor cellular uptake of PNAs in vitro, researche have successfully targeted a number of othergenes with PNAs,such as the bovine papillomavirus E2 protein [9], Ha-ras [10], c-myc [11,12], and telomerase [13]. For experiments involving antisense strategies, there ar several advantages to using PNAs as opposed to traditiona oligonucleotides. PNAs are electrically neutral oligomers, * Corresponding author. Tel.: 11-904-953-6902; fax: 11-904-953- 7117. E-mail address: mcmahon.beth@mayo.edu (B.M. Tyler-McMahon). Abbreviations: DAT, dopamine transporter; and PNA, peptide nucleic acid. Biochemical Pharmacology 62 (2001) 929 –932 0006-2952/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 0 6 - 2 9 5 2 ( 0 1 ) 0 0 6 9 8 - 0