INSERTION MUTATION AT THE C-TERMINUS OF THE SEROTONIN TRANSPORTER DISRUPTS BRAIN SEROTONIN FUNCTION AND EMOTION-RELATED BEHAVIORS IN MICE S. ZHAO, a * J. EDWARDS, a J. CARROLL, b L. WIEDHOLZ, c R. A. MILLSTEIN, c C. JAING, a D. L. MURPHY, b T. H. LANTHORN a AND A. HOLMES c a Lexicon Genetics Incorporated, 8800 Technology Forest Place, The Woodlands, TX 77381, USA b Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA c Section on Behavioral Science and Genetics, Laboratory for Integra- tive Neuroscience, National Institute on Alcohol Abuse and Alcohol- ism, National Institutes of Health, Rockville, MD 20852, USA Abstract—The 5-hydroxytryptamine transporter (5-HTT) regu- lates 5-hydroxytryptamine (5-HT) neurotransmission by remov- ing 5-HT from the synaptic cleft. Emerging evidence from clin- ical and genetic studies implicates the 5-HTT in various neuro- psychiatric conditions, including anxiety and depression. Here we report that a 5-HTT null mutant mouse line was generated by gene trapping that disrupted the sequence encoding the C- terminus of 5-HTT. This mutation resulted in significant reduc- tion of 5-HTT mRNA and loss of 5-HTT protein. Brain levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid, were markedly decreased in C-terminus 5-HTT / mice, while 5-HT uptake or 5-HT content in platelets was absent. Behavioral phe- notyping showed that C-terminus 5-HTT / mice were normal on a screen for gross behavioral, neurological, and sensory functions. In the tail suspension test for depression-related behavior, C-terminus 5-HTT / mice showed increased immo- bility relative to their / controls. By comparison, a previously generated line of 5-HTT / mice lacking exon 2, encoding the N-terminus of the 5-HTT, showed abnormally high immobility in response to repeated, but not acute, exposure to the tail sus- pension test. In a novel, brightly-lit open field, both C-terminus 5-HTT / mice and N-terminus 5-HTT / mice displayed decreased center time and reduced locomotor activity com- pared with their / controls. Both mutant lines buried signif- icantly fewer marbles than their / controls in the marble burying test. These findings further demonstrate the neurobio- logical functions of the 5-HTT and add to a growing literature linking genetic variation in 5-HTT function with emotional abnormalities. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: serotonin transporter, 5-HTT, SERT, knockout, be- havior, emotion. The 5-hydroxytryptamine transporter (5-HTT) belongs to a family of Na + /Cl - -dependent transporters that translocate biogenic amines through the plasma membrane. Clear- ance of 5-hydroxytryptamine (5-HT) from the synaptic cleft by the 5-HTT provides a critical regulatory mechanism controlling 5-HT-mediated neurotransmission. 5-HTT func- tion is in turn regulated by a variety of factors that presently remain poorly understood, such as signal transduction pathways affecting transport capacity via modulation of trafficking and localization of the protein to the cell surface (Blakely and Bauman, 2000). An increasing body of evidence implicates dysfunction of 5-HTT in mood and anxiety disorders. Drugs that target the 5-HTT, such as the 5-HT reuptake inhibitors, demon- strate clinical efficacy as antidepressants and anxiolytics (Ballenger, 1999; Kugaya et al., 2003). In addition, drug- free patients with mood and anxiety disorders exhibit a significant reduction in brain 5-HTT binding (Briley et al., 1980; Nemeroff et al., 1994; Malison et al., 1998; Willeit et al., 2000; Arango et al., 2001). More recent evidence has shown that functional gene variants in the 5-HTT are as- sociated with greater risk for mood and anxiety disorders. A common 44-base pair insertion/deletion polymorphism in the HTT (SLC6A4) which results in reduced 5-HTT mRNA level, 5-HTT binding, and 5-HT reuptake (Lesch et al., 1996; Little et al., 1998; Greenberg et al., 1999), has been associated with increased negative emotionality (for re- view, see Hariri and Holmes, in press). The role of the 5-HTT in mediating emotional behavior has previously been studied in mutant mice lacking a func- tional 5-HTT (Bengel et al., 1998; Lanfumey et al., 2000; Holmes et al., 2003b; Murphy et al., 2003; Li et al., 2004). In these mice, exon 2 of the htt gene was deleted via homolo- gous recombination. Exon 2 contains the start codon of the htt gene and encodes the N-terminus and first transmem- brane domain of the 5-HTT, a domain critical for 5-HT- and 5-HTT-antagonist binding and ion permeation (Barker et al., 1999). These “N-terminus” 5-HTT -/- mice provide a model of constitutive htt inactivation. In the present study, we further investigated the consequences of disrupting the 5-HTT in mice by a gene-trapping method that disrupted the C-termi- *Corresponding author. Tel: +1-281-863-3071; fax: +1-281-863-8088. E-mail address: szhao@lexgen.com (S. Zhao). Abbreviations: ES, embryonic stem; FST, forced swim test; -gal, -galactosidase; IRES, internal ribosomal entry site; LTR, long-termi- nal repeat sequences; NET, norepinephrine transporter; NIH, National Institutes of Health; pA, polyadenylation sequence; PB, phosphate buffer; Pgk, phosphoglycerate kinase-1 promoter; PKC, protein kinase C; PRP, platelet-rich plasma; Puro, puromycin N-acetyltransferase gene; QPCR, quantitative polymerase chain reaction; RT-PCR, re- verse transcriptase polymerase chain reaction; SA, splice acceptor; SD, splice donor; TBST, Tris-buffer saline containing 0.05% Tween- 20; TST, tail suspension test; 3= RACE, 3= rapid amplification of the cDNA ends; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryp- tamine (serotonin); 5-HTT, 5-hydroxytryptamine transporter (serotonin transporter). Neuroscience 140 (2006) 321–334 0306-4522/06$30.00+0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.01.049 321