Norepinephrine Transporter: A Candidate Gene for Initial Ethanol Sensitivity in Inbred Long-Sleep and Short-Sleep Mice Heather M. Haughey, Alan L. Kaiser, Thomas E. Johnson, Beth Bennett, James M. Sikela, and Nancy R. Zahniser Background: Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. Methods: In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol- induced loss of righting reflex (LORR) in ILS and ISS mice. Results: We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [ 3 H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [ 3 H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol- induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44 – 47 cM), compared with ISS homozygous strains. Conclusions: This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains. Key Words: Norepinephrine transporter, Loss of righting reflex, Alcohol sensitivity, Genetics, Quanti- tative trait loci. INTRODUCTION A LCOHOL DEPENDENCE, OR “alcoholism,” affects approximately 14 million Americans. Vulnerability to alcoholism is influenced by both environmental and genetic determinants; the genetic component, as estimated from adoption and twin studies, is between 40 and 60% (Enoch and Goldman, 2001; Han et al., 1999; Kendler et al., 1994; Prescott and Kendler, 1999). A low response to alcohol is a significant predictor of future alcohol abuse (Schuckit, 1994) and is genetically specified in both humans (Heath and Martin, 1992; Madden et al., 1995) and animals (Bald- win et al., 1991; Li, 2000; Moore et al., 1998; Schuckit et al., 1994; Schuckit and Smith, 1996). Genes influencing alco- holism remain elusive, although a number of candidate genes for alcohol dependence are under investigation (Dick and Foroud, 2003; Tyndale, 2003). Ethanol alters multiple excitatory, inhibitory, and neuro- modulatory neurotransmitter systems (for review, see Nevo and Hamon, 1995). For example, acute ethanol application alters release, clearance, and turnover rates of norepinephrine (NE), dopamine, and serotonin. NE is a ubiquitous neuro- transmitter whose actions are dynamically regulated by the NE transporter (NET) (Xu et al., 2000). NE influences atten- tion, mood, motivation, stress, thermoregulation, and reward (Cooper et al., 2003; Nestler et al., 2001; Weinshenker et al., 2002). Alterations in NE neurotransmission play a critical role in pathologic conditions such as depression and drug abuse and may contribute to alcoholism. Supporting this, Weinsh- enker and colleagues (2000) found that dopamine -hydroxylase knockout mice (Dbh -/- ), which lack NE, are supersensitive to the sedative effects of ethanol, and this effect is blocked by acute replacement of central NE, prior to eth- anol administration. From the Department of Pharmacology (HMH, ALK, JMS, NRZ) and the Neuroscience Program (JMS, NRZ), University of Colorado Health Sciences Center, Aurora, Colorado; and the Institute for Behavioral Genetics (TEJ, BB), University of Colorado, Boulder, Colorado. Received for publication Jan 21, 2005; accepted for publication Jul 5, 2005 Supported in part by NIAAA grants P50 AA03527 (NRZ), RO1 AA11853 (JMS), RO1 AA08940 (TEJ), RO1 AA011984 (TEJ), and KO4 AA00195 (TEJ) and by NIDA grants K05 DA15050 (NRZ) and F32 DA14476 (HMH). Reprint requests: Heather M. Haughey, Ph.D., University of Colorado at Boulder Department of Psychology, C-345, Boulder, CO 80309 – 0345; Fax: 303-492-2967; E-mail: heather.haughey@colorado.edu. Copyright © 2005 by the Research Society on Alcoholism. DOI: 10.1097/01.alc.0000183009.57805.a6 0145-6008/05/29010-1759$03.00/0 ALCOHOLISM:CLINICAL AND EXPERIMENTAL RESEARCH Vol. 29, No. 10 October 2005 Alcohol Clin Exp Res, Vol 29, No 10, 2005: pp 1759–1768 1759