Note: Outstanding Young Investigator Awardee, 7th Annual Meeting of the International Behavioural and Neural Genetics Society, Sitges, Spain, June 9–12, 2005.
The type 1 equilibrative nucleoside transporter regulates anxiety-like behavior in mice
Article first published online: 13 FEB 2007
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd
Genes, Brain and Behavior
Volume 6, Issue 8, pages 776–783, November 2007
How to Cite
Chen, J., Rinaldo, L., Lim, S.-J., Young, H., Messing, R. O. and Choi, D.-S. (2007), The type 1 equilibrative nucleoside transporter regulates anxiety-like behavior in mice. Genes, Brain and Behavior, 6: 776–783. doi: 10.1111/j.1601-183X.2007.00311.x
- Issue published online: 13 FEB 2007
- Article first published online: 13 FEB 2007
- Received 14 August 2006, revised 31 January 2007, accepted for publication 3 February 2007
- behavioral genetics;
- nucleoside transporter;
- psychiatric disorders
Activation of adenosine receptors in the brain reduces anxiety-like behavior in animals and humans. Because nucleoside transporters regulate adenosine levels, we used mice lacking the type 1 equilibrative nucleoside transporter (ENT1) to investigate whether ENT1 contributes to anxiety-like behavior. The ENT1 null mice spent more time in the center of an open field compared with wild-type littermates. In the elevated plus maze, ENT1 null mice entered more frequently into and spent more time exploring the open arms. The ENT1 null mice also spent more time exploring the light side of a light–dark box compared with wild-type mice. Microinjection of an ENT1-specific antagonist, nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside), into the amygdala of C57BL/6J mice reduced anxiety-like behavior in the open field and elevated plus maze. These findings show that amygdala ENT1 modulates anxiety-like behavior. The ENT1 may be a drug target for the treatment of anxiety disorders.
Adenosine suppresses neurotransmitter release, reduces neuronal excitability and regulates ion channel function through activation of four classes of G protein-coupled receptors, A1, A2A, A2B and A3, in the central nervous system (Dunwiddie & Masino 2001). Antagonism of A1 and A2A receptors appears to be responsible for the stimulant effects of adenosine receptor antagonists such as caffeine (Fredholm et al. 1999). Despite their widespread distribution in the brain, little is known about the physiological responses mediated by A2B and A3 receptors.
Adenosine analogues are anxiolytic (Dunwiddie & Masino 2001). In mice, the selective A1 receptor agonist, N6-cyclopentyladenosine (CPA), reduces anxiety-like behavior and inhibits the anxiogenic effect of caffeine (Jain et al. 1995). In contrast, caffeine, a nonselective adenosine receptor antagonist, and CGS15943A, a mixed A1/A2A adenosine receptor antagonist, are anxiogenic in animals and humans (Baldwin & File 1989; Griebel et al. 1991).
A major function of nucleoside transporters is to control intracellular and extracellular concentrations of adenosine in the brain (Dunwiddie & Masino 2001). Nucleoside transport plays an important role in salvaging extracellular nucleosides for intracellular nucleotide synthesis and for regulating endogenous nucleoside levels. Two main plasma membrane transporter families have been characterized. The sodium-independent equilibrative transporters [equilibrative nucleoside transporter (ENT)] mediate nucleoside transport bidirectionally depending on the concentration gradient across the plasma membrane. Under normal physiological conditions, this is higher outside cells, leading to influx of adenosine through ENTs (Dunwiddie & Masino 2001). Sodium-dependent concentrative transporters mediate an inwardly directed transport driven by the sodium electrochemical gradient (Anderson et al. 1999a). Four ENT subtypes have been cloned (Baldwin et al. 2004). Type 1 ENT1 (ENT1) is sensitive to nanomolar concentrations of nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside; NBMPR), whereas ENT2 is resistant to NBMPR up to 1 mm (Crawford et al. 1998; Griffiths et al. 1997a,b; Yao et al. 1997). The ENT1 and ENT2 are widely expressed in the central nervous system (Anderson et al. 1999b; Jennings et al. 2001), while ENT3 appears to be expressed outside the nervous system (Hyde et al. 2001) and mainly intracellularly, where it colocalizes with lysosomal markers in cultured human cells (Baldwin et al. 2005). Expression and pharmacological properties of ENT4 have yet to be fully characterized (Baldwin et al. 2004). Subtypes ENT1 and ENT2 are about 50% homologous in amino acid sequence and contain 11 putative transmembrane domains.
We recently generated mutant mice that lack ENT1 and studied their behavioral responses to ethanol (Choi et al. 2004) because cell culture studies have shown that ethanol inhibits ENT1 and this action is responsible for stimulation of adenosine signaling by ethanol in vitro (Nagy et al. 1990). We found, indeed, that ENT1 null mice are less sensitive to the intoxicating effects of ethanol when compared with wild-type littermates, supporting a role for ENT1 and adenosine in acute effects of ethanol in vivo (Choi et al. 2004). Because adenosine also regulates anxiety, we performed the present study to investigate the role of ENT1 in modulation of anxiety by examining anxiety-like behaviors in ENT1 null mice and in C57BL/6J mice treated with a selective inhibitor of ENT1 microinjected into the amygdala.
Materials and methods
The ENT1 null mice were generated on a C57BL/6J × 129X1/SvJ background using the targeting vector pKSloxPNT-mod (kindly provided by A. L. Joyner, New York University, NY) to delete coding exons 2 and 4, as described (Choi et al. 2004). Chimeric mice were bred with C57BL/6J mice to generate F1 hybrids. These ENT1 heterozygous mutant F1 mice were intercrossed to generate F2 hybrid (∼50% C57BL/6J and ∼50% 129X1/SvJ) littermates for experiments. For microinjection studies, C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in standard Plexiglas cages with rodent chow and water available ad libitum. The colony room was maintained on a 12-h light/dark cycle with lights on at 0700 h. For all experiments, we used male mice when they reached approximately 10 weeks of age. We used different mice for each experiment. Animal care and handling procedures were approved by the Gallo Center and the Mayo Clinic Institutional Animal Care and Use Committees in accordance with National Institutes of Health guidelines.
Spontaneous locomotor activity was measured in open-field chambers (41 × 41 cm). Mice were examined in a quiet room under normal fluorescent room light (500 lux). Mice were handled and weighed daily for 1 week prior to activity testing. On the test day, mice were weighed and placed immediately in the activity chambers. Horizontal distance traveled (cm) was recorded for 1 h using a video-tracking system (ViewPoint Life Sciences Inc., Montreal, Canada). For assessment of activity in the center of the field, the chamber floor was divided post hoc into a central zone (21 × 21 cm; center equidistant from all four walls of the chamber) and a peripheral zone (the remaining area of the floor). Distance traveled and time spent in each area were calculated from the locomotor activity data. After each test session, the equipment was cleaned with 70% ethanol to remove animal odors.
Elevated plus maze
The elevated plus maze test has been validated for measurement of anxiety-like behavior in rodents (Lister 1987; Pellow et al. 1985). Mice were examined in a quiet room under normal fluorescent room light (500 lux). The maze was elevated 60 cm off the floor, constructed of wood coated with gloss enamel white paint, having two open arms (37 × 8 cm) and two closed arms (37 × 8 × 14 cm) extending from a common central platform (8 × 8 cm). An observer blind to the genotype of the mouse placed it on the central platform and allowed it to explore for 5 min. The observer remained in the room during the test. The x and y co-ordinates of the center of the mouse were continuously monitored (30 Hz) in the maze using a video-tracking system (ViewPoint Life Sciences). We scored (1) percentage of total time spent in the open arms, (2) percentage of entries to the open arms, (3) number of entries into the closed arms and (4) total distance traveled in the maze. We ignored time spent in the center and calculated the percentage of open time and open entries as [open/(open + closed) × 100]. Following each session, the maze was cleaned with 70% ethanol to remove odors.
The light–dark box test measures anxiety-like behavior by contrasting the tendency of mice to explore a novel environment with the aversive properties of a brightly lit open field (Crawley & Goodwin 1980). An illuminated (22 lux) open-field chamber (28 × 27 × 20 cm) located in a sound-attenuating cubicle was equipped with an insert (14 × 27 × 20 cm) that made half of the area dark (2 lux) (Med Associates, Georgia, VT, USA). An arched opening (5 × 7 cm) allowed the mouse to pass freely between the two halves of the chamber. The mouse was placed in a back corner of the light side and tested for a 5-min period. The following behaviors were scored: (1) latency to enter the dark side, i.e. the delay before the mouse entered with all four paws into the dark side for the first time, (2) latency to re-enter the light side, (3) the total time spent in the light side, (4) distance traveled in light side, (5) number of crossings between light and dark sides and (6) total distance traveled. Following each session, the maze was cleaned with 70% ethanol to remove odors.
To examine whether inhibition of ENT1 in the amygdala or in the dorsal striatum alters anxiety-like behavior, we microinjected C57BL/6J male mice (Jackson Laboratory) with the ENT1-specific antagonist, NBMPR. Animals were anesthetized with ketamine/xylazine (100 and 15 mg/kg, i.p.; Sigma-Aldrich Co., St Louis, MO, USA) and placed in a digital stereotaxic alignment system (Model 1900, David Kopf Instruments, Tujunga, CA, USA). After the skull was exposed, the mouse was positioned with bregma at the focal point, and the skull was leveled using a dual-tilt measurement tool. At the desired co-ordinates, 0.5-mm holes were drilled for placement of stainless steel guide cannulas (26 gauge; Plastics One Inc., Roanoke, VA, USA) aimed 1 mm above the central nucleus of the amygdala (co-ordinates: antero-posterior (AP) −1.06 mm, medio-lateral (ML) ±2.2 mm and dorso-ventral (DV) −3.8 mm) or caudate-putamen (CPu) (co-ordinates: AP 0.5 mm, ML ±2.2 mm and DV −2.8 mm) (Franklin & Paxinos 2001). The guide cannulas were fixed to screws in the skull with dental cement, and dummy cannulas were inserted to prevent clogging. The dummy cannulas extended 1 mm past the tips of the guide cannulas. The mice were allowed to recover from anesthesia on a heated pad for 1 h and were left undisturbed in their home cage for 10–12 days prior to testing.
For microinjection experiments, dummy cannulas were removed and infusion cannulas (33 gauge, Plastics One Inc.) were immediately inserted; like the dummy cannulas, the tips of the infusion cannulas also extended 1 mm past the tips of the guide cannulas (Zhao & Davis 2004). Injections were given 15 min before behavioral testing. Nitrobenzylmercaptopurine riboside [50 μm, dissolved in saline containing 2% dimethyl sulphoxide (DMSO); Sigma-Aldrich Co.] or 2% DMSO (vehicle control) was infused using an infusion pump (Harvard Apparatus, Holliston, MA, USA) at 0.3 μl/min in a volume of 0.5 μl per side. The infusion cannulas were left in place for 1 min to allow for diffusion.
At the end of the experiment, mice were perfused intracardially with 4% paraformaldehyde, and the brains were removed. Cannula location was verified histologically by haematoxylin and eosin (H & E) staining. Only mice that showed cannula placement in the amygdala were analyzed.
All data are presented as mean ± SEM. For open-field locomotor testing, data were analyzed by three-way repeated measures analysis of variance (anova) with between-subject factors for genotype and gender, and a repeated measure for test day. This analysis was followed by a Tukey’s post hoc test for individual comparisons. All other data were analyzed by two-tailed t-tests. Results were considered significantly different where P < 0.05.
Reduced anxiety-like behavior in ENT1 null mice
We examined spontaneous locomotor activity in open-field chambers for 1 h per day over 3 consecutive days using 10 male and 5 female mice of each genotype. Three-way anova with factors for gender and genotype, and a repeated measure for day, showed a significant effect of day (F2,78= 3.85, P = 0.026) but no effect of genotype (F1,78= 0.61, P = 0.43) or gender (F1,78= 1.23, P = 0.27); there were also no interactions between day and genotype or gender. Because there was no effect of gender, we combined data from male and female mice (Fig. 1a). Analysis of these combined data by two-way anova showed a significant effect of day (F2,84= 4.76, P = 0.01), but no effect of genotype (F1,84= 1.15, P = 0.29) and no interaction between genotype and day (F1,84= 0.19, P = 0.82). These results indicate that ENT1 null mice show normal locomotor activity and habituate normally to the open-field chamber.
Because mice have a natural aversion to the brightly lit center of an open field, comparison of activity in the center vs. the periphery of the field gives an indication of anxiety-like behavior (Crawley 2000). Analysis of data for locomotor activity in the center of the open field by three-way anova showed a main effect of genotype (F1,78= 4.28, P = 0.042) without interaction with other factors. Again, there was no main effect of gender (F1,78= 0.32, P = 0.57) or gender by genotype interaction (F1,78= 0.31, P = 0.58), so we combined data from male and female mice (Fig. 1b). Two-way anova of these combined data confirmed a significant effect of genotype (F1,82= 5.30, P = 0.02) without a main effect of test day (F2,82= 0.53, P = 0.59) or an interaction between genotype and test day (F2,82= 0.11, P = 0.89). These findings indicate that ENT1 null mice show less aversion than wild-type mice to the center of the open field, which is consistent with decreased anxiety-like behavior. Because these results did not show an effect of gender, we combined data from male and female mice in subsequent studies.
We next performed an elevated plus maze test, which exploits the conflict between the desire to explore a novel area and aversion to open areas and height (Crawley 2000; Gordon & Hen 2004). The ENT1 null mice spent a greater percentage of time in the open arms (Fig. 2a) and made a greater percentage of visits into the open arms (Fig. 2b) compared with wild-type mice. There was no difference in the number of entries into the closed arms (Fig. 2c) or in total distance traveled (Fig. 2d), indicating that locomotor activity was similar in both genotypes. These findings further indicate that deficiency in ENT1 reduces anxiety-like behavior.
Finally, we examined anxiety-like behavior using a light–dark shuttle box, which examines the conflict between the drive to explore and aversion to a brightly lit space (Crawley & Goodwin 1980). Compared with wild-type littermates, ENT1 null mice showed a trend toward an increased latency to enter the dark side of the box (Fig. 3a), but this difference was not significant. However, ENT1 null mice displayed a significantly shorter latency to re-enter the light side (Fig. 3b), and spent more time (Fig. 3c) and traveled a greater distance (Fig. 3d) in the light side of the box compared with wild-type mice. These findings further indicate that anxiety-like behavior is reduced in ENT1 null mice. There was no difference among the genotypes in the number of crossings between light and dark sides of the box (Fig. 3e) or in the total distance traveled (Fig. 3f), again confirming that locomotor activity is similar in both genotypes.
Inhibition of ENT1 in the amygdala, but not in the dorsal striatum, reduces anxiety-like behavior
The decreased anxiety-like behavior that we observed in ENT1 null mice could have resulted from absence of ENT1 signaling in the adult brain or during development, or the presence of a 129X1/SvJ allele of a gene near the ENT1 locus in the ENT1 null population. Because the absence of ENT1 is systemic in these mice, we also do not know from our results which brain regions are responsible for their reduced anxiety-like behavior. We reasoned that if this phenotype is due to absence of ENT1 in a specific region of the adult brain, then we should be able to phenocopy the mutant behavior by administration of an ENT1 antagonist into the critical brain region.
Because the amygdala regulates anxiety (Wright et al. 1996), and ENT1 is expressed highly in the amygdala (Anderson et al. 1999b), we examined the role of amygdala ENT1 by microinjecting the potent, ENT1-selective adenosine uptake inhibitor, NBMPR, into that brain region in C57BL/6J mice. We confirmed cannula placement by histological examination (Fig. 4a). On the first day of the open-field test, the total distance traveled was similar between vehicle- and NBMPR-treated groups (Fig. 4b), indicating the inhibition of ENT1 in the amygdala does not influence the locomotor activity in the open field. However, the NBMPR-injected group spent more time in the central area of the open field compared with the vehicle-treated group (Fig. 4c), consistent with a reduction in anxiety-like behavior following NBMPR injection. Vehicle-treated C57BL/6J mice traveled a greater total distance in the open field compared with F2 hybrids (Fig. 1a). This difference is likely due to different genetic backgrounds (Crabbe et al. 1999).
In the elevated plus maze test, the NBMPR-treated group spent a significantly greater percentage of time in the open arms (Fig. 4d), but there was no significant difference in the percentage of open-arm entries between vehicle- and NBMPR-treated groups (Fig. 4e). Mice treated with NBMPR also exhibited fewer entries into the closed arms (Fig. 4f) and traveled a shorter total distance than did the vehicle-treated group (Fig. 4g). Thus, NBMPR reduced anxiety-like behavior on one measure in the plus maze, while it also depressed exploratory activity in the maze.
To examine whether inhibition of ENT1 in the amygdala is specific for decreasing anxiety-like behavior, we injected NBMPR into the CPu, which expresses ENT1 (Anderson et al. 1999b; Jennings et al. 2001) and is involved in motor function, but also makes connections with the amygdala (Wright et al. 1996). We confirmed cannula placement as shown in Fig. 5(a). We found that inhibition of dorsal striatal ENT1 did not alter the percentage of distance in the central area of the open field or the total distance traveled in the field (Fig. 5b,c). Similarly, in the elevated plus maze test, intra-CPu injection of NBMPR did not affect the percentage of time spent (Fig. 5d) or the percentage of entries into the open arms (Fig. 5e). The number of entries into the closed arms (Fig. 5f) and the total distance traveled (Fig. 5g) were also similar between the groups after CPu injection of NBMPR. These data show that ENT1 in the amygdala, rather than in the Cpu, contributes to anxiety-like behavior.
The results of the present study show that the absence of ENT1 is associated with reduced anxiety-like behavior in mice. Microinjection of NBMPR, a specific ENT1 inhibitor, into the amygdala of C57BL/6J mice also reduced anxiety-like behavior in the open field and in the elevated plus maze. This finding indicates that the phenotype of reduced anxiety-like behavior in ENT1 null mice is due to absence of ENT1 in the adult brain and is not due to a developmental change or to effects of a 129X1/SvJ allele of a gene near the ENT1 locus. Furthermore, this result suggests an important role for amygdala ENT1 in the regulation of anxiety-like behavior.
The effect of gene deletion appeared to be more robust than NBMPR injection in our elevated plus maze studies. This may be due to differences in genetic background between the mice used in these different experiments. For example, in wild-type C57BL/6J × 129X1/SvJ hybrid mice, about 30% of arm entries were into the open arms, whereas in saline-treated C57BL/6J mice, 45% of arm entries were into the open arms. The greater percentage of open-arm entries in C57BL/6J mice may have limited our ability to detect a further increase induced by an anxiolytic effect of NBMPR. Alternatively, the limited anxiolytic response to NBMPR may have been due to incomplete inhibition of amygdala ENT1 by our microinjections or may suggest that regions other than the amygdala are also involved in the effect of ENT1 on anxiety-like behavior. However, microinjection of NBMPR into the CPu had no effect on behavior both in open field and in the elevated plus maze, indicating that the effect of NBMPR, by comparison, was specific for the amygdala.
Injection of NBMPR into the amygdala also decreased the number of closed-arm entries and total distance traveled in the elevated plus maze. This decrease in locomotor behavior was not associated with episodes of freezing and was not likely due to a sedative effect because a similar concentration of NBMPR did not decrease the distance traveled in the open field following amygdala injection. Moreover, NBMPR injection into the CPu, which regulates locomotor activity, did not alter behavior in the elevated plus maze. Given recent evidence for amygdala involvement in incentive processes (Balleine & Killcross 2006), our observations suggest a role for ENT1 in motivation to explore the maze.
As ENT1 regulates adenosine levels in the brain, the decreased anxiety-like behavior we observed is likely mediated by adenosine receptor signaling. The selective A1 receptor agonist, CPA, is anxiolytic at 10–50 mg/kg, whereas the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, has no significant effect on anxiety-related behavior, but blocks the anxiolytic effect of CPA (Jain et al. 1995). A2A receptor drugs do not alter anxiety-like behaviors, indicating that A1 receptors mediate the anxiolytic properties of adenosine. By contrast, A2A receptor null mice show increased anxiety-like behavior (Ledent et al. 1997), which may reflect a change during development or a compensatory response to lifelong absence of A2A receptors. However, high doses of caffeine, a nonselective adenosine receptor antagonist, are anxiogenic in animals and humans (Fredholm et al. 1999), and a polymorphism in the human A2A receptor, 1976T C in the 3′ untranslated region that may alter the A2A receptor expression is associated with individual differences in the anxiogenic response to caffeine (Alsene et al. 2003) and in susceptibility to panic disorder (Hamilton et al. 2004). Therefore, A2A receptors may contribute to anxiety, particularly if there are long-standing deficits in A2A receptor expression or function.
Because deletion of ENT1 or microinjection of NBMPR reduced anxiety-like behavior, it appears that ENT1 normally acts to reduce levels of extracellular adenosine in the amygdala and that absence or inhibition of ENT1 elevates extracellular adenosine in that brain region. This is different from our prior studies showing that ENT1 null mice have reduced adenosine tone in the striatum, resulting in increased release of glutamate due to reduced activation of presynaptic A1 receptors (Choi et al. 2004). It is technically very difficult to measure extracellular adenosine levels directly because of the rapid metabolism of adenosine (half-life = 10–30 seconds), although some studies have been done by in vivo microdialysis (Ballarin et al. 1991). Recently, a more sensitive and direct method has been developed for in vitro measurements (Frenguelli et al. 2003), and will hopefully be successfully adapted to allow us to accurately measure regional brain levels of extracellular adenosine in the future.
The ENT1 plays an important role in ethanol intoxication (Choi et al. 2004). Ethanol inhibits adenosine uptake through ENT1, resulting in increased levels of extracellular adenosine (Nagy et al. 1990), which mediates several effects of ethanol through stimulation of A1 adenosine receptors (Dar 1997, 2001). A1 receptors also play an important role in anxiety associated with alcohol withdrawal as A1 agonists decrease withdrawal symptoms such as tremors and seizures (Concas et al. 1994; Kaplan et al. 1999) and anxiety-like behavior (Prediger et al. 2006). Alcohol-induced anxiety is of particular clinical interest because anxiety disorders are often associated with alcohol dependence (Kessler et al. 1994), and excessive anxiety can promote high levels of alcohol consumption (Kushner et al. 2000; Schuckit et al. 1997). However, our findings with ENT1 null mice indicate that increases in anxiety-like behavior and alcohol consumption can be genetically dissociated because ENT1 null mice show reduced anxiety-like behavior yet drink more alcohol than wild-type littermates (Choi et al. 2004).
In conclusion, our results indicate that the absence or inhibition of ENT1 can reduce anxiety-like behavior in mice. The ENT1 regulation of anxiety most likely involves alterations in adenosine signaling within the amygdala. Strategies to inhibit ENT1 in the brain may prove useful to treat excessive anxiety in humans.
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We are grateful to S. Taylor and J. Connolly for mouse handling, and D. Frederixon for preparing the manuscript. This work was supported in part by funds provided by the State of California for Medical Research on Alcohol and Substance Abuse through the University of California at San Francisco (R. O. M.), by the Samuel Johnson Foundation for Genomics of Drug Addiction Program at Mayo Clinic (D. -S. C) and by the National Institutes of Health grants AA013588 (to R. O. M.), and AA015164 (to D. -S. C).