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Keywords:

  • Knockout;
  • locomotor activity;
  • MAO-B;
  • nicotine;
  • open field

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Low levels of monoamine oxidase-B (MAO-B) activity, such as those observed in smokers, are also associated with behavioral traits such as a heightened responsiveness to novelty. However, the exact mechanism by which low MAO-B activity influences smoking and heightened responsiveness to novelty is still poorly understood. We used MAO-B knockout (KO) mice to test the hypothesis that MAO-B concomitantly affects locomotor responses in a novel inescapable open field and nicotine intake. Male wild-type (WT) and MAO-B KO mice were placed in an inescapable open field and their horizontal locomotor activity was measured for 30 min per day for 5 days. MAO-B KO mice exhibited impaired within-session habituation of locomotor activity, as compared to WT mice. Separate groups of male WT and MAO-B KO mice were individually housed in their home cages with two water bottles. One of the bottles contained tap water and the other contained nicotine (0, 3.125, 6.25, 12.5, 25, 50 or 100 µg/ml). The total amount of water and nicotine solution consumed was measured every three days for 16 days. MAO-B KO mice and WT mice consumed equal amounts of nicotine and exhibited comparable concentration-dependent nicotine preference and aversion over a period of 16 days. The data suggest that the absence of MAO-B impairs the ability of mice to habituate in the inescapable environment, but does not alter their nicotine intake.

It has been established that genetic variations can influence the likelihood of being a smoker (Heath et al. 1999; Kendler et al. 1999; Madden et al. 1999; True et al. 1999). A number of genes have been proposed to contribute to heightened susceptibility to smoking, including the genes encoding monoamine receptors and associated enzymes (Arinami et al. 2000; Lerman & Berrettini 2003; Walton et al. 2001).

Differences in monoamine oxidase-B (MAO-B) activity of up to 30-fold have been reported across different individuals (Murphy et al. 1976). These differences are due, at least in part, to genetic polymorphisms. A polymorphism within intron 13 correlates with MAO-B enzyme activity in platelets (Garpenstrand et al. 2000) and in the frontal cortex of humans (Balciuniene et al. 2002); the A allele of intron 13 is associated with higher MAO-B activity than is the G allele in the frontal cortex (Balciuniene et al. 2002). Moreover, AP-2β, a transcription factor that regulates MAO-B gene expression, also contains an allele that is associated with low MAO-B activity in platelets (Damberg et al. 2000).

Inherent levels of MAO-B are thought to underlie broad behavioral traits. Individuals with low levels of MAO-B activity in platelets exhibit traits variously labeled as ‘novelty seeking’, ‘sensation seeking’, ‘experience seeking’ and ‘impulsivity’ (see Oreland et al. 2002; Zuckerman & Kuhlman 2000). These behavioral traits have been found to be associated with smoking status (Heath et al. 1995; Kopstein et al. 2001; Wills et al. 1994; Zuckerman & Kuhlman 2000), as well as with the initial response to nicotine in non-smokers (Perkins et al. 2000). These comorbid traits represent predisposing factors for smoking, rather than a consequence of smoking, because they precede the initiation of smoking (Lipkus et al. 1994; Masse & Tremblay 1997; Sher et al. 2000). Although it has been established that compounds other than nicotine in tobacco smoke alter MAO-B activity (Hauptmann & Shih 2001; Khalil et al. 2000; see also Fowler et al. 2003), it remains unclear whether inherently low levels of MAO-B activity causally affect both novelty response and nicotine addiction. Studies in humans have not fully delineated the genetic mechanisms underlying the relationship between smoking and novelty response.

Animal studies have also shown that individual differences in locomotor activity in a novel, inescapable open field can predict the reinforcing effects of amphetamine, cocaine, morphine, ethanol and nicotine in rodents (Ambrosio et al. 1995; Bevins et al. 1997; Cailhol & Mormede 1999; Dellu et al. 1996; Deroche et al. 1993; Elmer et al. 1995; Grimm & See 1997; Hiroi et al. 1997; Hooks et al. 1991; Klebaur & Bardo 1999; Klebaur et al. 2001; Mantsch et al. 2001; Marinelli & White 2000; Nadal et al. 2002; Piazza et al. 1989, 1990, 2000; Pierre & Vezina 1997; Suto et al. 2001). However, the genetic basis for this association is poorly understood. We hypothesized that the MAO-B gene concomitantly contributes to the regulation of locomotor activity and nicotine intake. We directly tested this hypothesis by examining locomotor activity in an inescapable novel environment and nicotine intake in the two-bottle nicotine intake method in MAO-B knockout (KO) mice.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Animals

We used male MAO-B KO and wild-type (WT) mice at the age of 2–4 months. This mouse line was created by homologous recombination, as described previously (Grimsby et al. 1997). Briefly, MAO-B KO mice were created using ES cells derived from 129S6/SvEv mice. Chimeric mice that exhibited germline transmission were then mated with 129S6/SvEv mice, resulting in a homogeneous inbred strain background. Because our MAO-B KO and WT mice were a congenic line on an essentially homogeneous genetic background, we bred WT mice by WT-WT pairs and MAO-B KO mice by KO-KO pairs. Animals were experimentally naïve and were housed in groups of up to five per cage. They were maintained on a 14 h/10 h light/dark cycle under light conditions from 06:00 to 20:00 and had free access to food and water. All studies were carried out in accordance with the Guide for Care and Use of Laboratory Animals of the Albert Einstein College of Medicine.

Locomotor activity in an inescapable open field

We used 10 WT mice and 8 MAO-B KO mice for this analysis. Horizontal locomotor activity was measured as an index of locomotor activity in six 5-min bins for 30 min per day between 10:00 and 11:00 on each of 5 days. Mice were brought to a room adjacent to the test room at least 20 min prior to the beginning of testing each day. We used four sets of automated activity apparatuses made from transparent Plexiglass (26 cm × 26 cm × 38.5 cm, Truscan, Coulbourn Instruments, Allentown, PA). The apparatus was cleaned with 70% ethanol and rinsed with water after each session to remove any residual olfactory cues. The only source of illumination was the fluorescent light on the ceiling of the room. The level of illumination was approximately 97 lux at the center of the open field. The small size of the open field, the low level of illumination and the use of transparent walls were intended to minimize anxiety in performing the task (Archer 1973; Crawley 2000). This apparatus detects horizontal activity through a set of beams located 1.5 cm above the floor of the apparatus. The open field was divided into squares (1.5 cm × 1.5 cm). Each side had 16 beams, dividing the open field into 289 squares. The distance traveled was used as a measure of locomotor activity.

Oral nicotine intake

Oral intake is a suitable method for administering nicotine, as unlike systemic injection it mimics the frequent, low-level exposure to nicotine experienced by cigarette smokers (Rowell et al. 1983). Moreover, unlike minipumps or pellets that release nicotine continually, regardless of circadian rhythm, mice consume nicotine primarily during the active phase in the oral intake method, similar to cigarette smokers (Kotlus & Blizard 1998; Pietila et al. 1995). The oral nicotine intake method has been shown to result in nicotine blood levels in mice similar to those in cigarette smokers (Rowell et al. 1983).

Separate groups of experimentally naïve mice (n = 6–9/concentration/genotype) were used for this analysis. MAO-B KO mice and WT mice were individually housed in their home cages (28 cm × 17 cm × 12 cm) at the onset of the experiment. Food was available ad libitum. Two bottles were provided in each cage: one contained tap water with (-)- nicotine base (1.01 g/ml unit, Sigma/RBI; final concentrations were 0, 3.125, 6.25, 12.5, 25, 50 or 100 free base µg/ml) and the other contained tap water only. The concentration range used has been previously shown to elicit dose-dependent nicotine drinking behavior in mice (Adriani et al. 2002; Klein et al. 2003; Meliska et al. 1995a,b; Robinson et al. 1996) and to achieve reliable serum levels of nicotine and cotinine in mice (Adriani et al. 2002; Klein et al. 2003; Sparks & Pauly 1999). Alkaline nicotine free base, as opposed to its acidic counterpart, was used because it is readily absorbed through mucous membranes (Armitage & Turner 1970; Benowitz 1999; Rowell et al. 1983). Each animal was given a single concentration of nicotine. For the 0 mg/ml concentration, mice received two equivalent bottles of water. The weight of each bottle, as well as body weight, was assessed and fresh nicotine solution and water were given between 10:00 and 11:00 every three days for a total of 16 days. Thus, data were recorded on five days (i.e. days 4, 7, 10, 13 and 16). Bottle weight has been shown to be a reliable measure of fluid intake (Stolerman & Kumar 1972). Nicotine consumption was expressed as nicotine intake (mg)/body weight (kg)/3 days. Nicotine preference/aversion was expressed as a ratio of fluid intake from a nicotine bottle divided by the total fluid intake from the nicotine bottle and the water bottle.

A nicotine bottle was placed on the right and a water bottle was placed on the left side of the cage covers in all the groups. We did not alternate the positions of the nicotine and water bottles. In a pilot study, we placed one nicotine bottle and one water bottle in each cage of a group of WT mice. Mice exhibited changes in nicotine drinking in a concentration-dependent manner when the bottle containing nicotine was placed on the right side. In contrast, when a nicotine bottle was placed on the left side of the cage, the mice did not consume nicotine in a concentration-dependent manner as clearly as when the nicotine bottle was placed on the right side. To determine whether this effect was specific in any way to nicotine, we also conducted a pilot study in which a saccharin bottle was alternately placed on the right and left side. Saccharin preference was 15% higher when the position of a saccharin bottle was switched from the left to right in both WT and MAO-B KO mice. This is likely to reflect an animal's drinking history in their home cage prior to the introduction of the two bottles, rather than individual differences. Normally, mice are housed in a cage in which a single, regular water bottle is placed on the cage ceiling. In our colony, this side was on the right side of the cage ceiling. In fact, when two water bottles were introduced into this cage, mice preferred to drink water from the bottle on the right side, as compared to the left.

Most studies that constantly switch the position of a drug-containing bottle and a water bottle conceal this fluctuation by combining data across days. However, this procedure would generate data with unusually large levels of variation and significant differences among the groups could be obscured, thereby making it difficult to detect subtle differences. Moreover, the switched method would not allow analysis of daily fluctuations of nicotine drinking, as nicotine drinking in this method would reflect the position of a nicotine bottle rather than chronic effects of nicotine intake. Therefore, we decided to use the fixed position method so that even small differences could be detected.

Another reason that we chose the fixed position method is that this procedure, unlike the commonly used bottle-switching method which attempts to ‘break’ any pre-existing position preference, does not tax an animal's ability to discriminate the two positions and reverse a previously established response. This procedural modification was necessary because it has been reported that some KO mice have abnormalities in behavioral reversal. That is, animals have abnormalities in changing a response that is contingent upon reinforcement once the response is established. MAO-B KO mice exhibit facilitated behavioral reversal at the onset of reversal sessions in a water maze (Holschneider et al. 1999). Heyser et al. (2000) reported that DARPP-32 KO mice have difficulty in switching from pressing one lever to a new lever when reinforcement is removed from the original lever and switched to the new lever. Moreover, 5-HT1B KO mice exhibit a more rapid reversal than WT mice in a water maze (Buhot et al. 2003). If the positions of the nicotine bottles were switched, the data could reflect the ability of mice to discriminate the two positions and reverse responses, as well as the pharmacological effects of nicotine. Thus, in the switched method, it would be difficult to assess the pharmacological effects of nicotine without the confounding impact of an abnormal behavioral shift.

We monitored bottles for spillage for 4–5 days prior to testing. During this pretest monitoring period, spillage caused a visible wet spot below the tip of the bottle nozzles. We examined the bedding in each cage to determine whether a wet spot was present. When we noticed spillage during the pretest monitoring period, those bottles were not used during testing. No spillage was detected during testing.

Statistical analysis

Data were analyzed using three-way analysis of variance (anova). The minimum level of significance accepted was set at P < 0.05. For additional, multiple two-way anovas, the significance threshold was adjusted by Bonferroni's correction.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

MAO-B KO mice exhibit prolonged locomotor responses

Locomotor activity data were analyzed using a three-way anova, including genotype (WT vs. MAO-B KO), time interval (5–30 min, repeated measure) and day (Days 1–5, repeated measure). Overall, MAO-B KO mice showed higher levels of locomotor activity than WT mice, as evidenced by a significant main genotype effect (F1,16 = 6.22, P < 0.05) (see Fig. 1). Locomotor activity also significantly declined across time intervals (F5,80 = 46.70, P < 0.01) and across days (F4,64 = 11.44, P < 0.01). The higher levels of locomotor activity in MAO-B KO mice, as compared to WT mice, exhibited a significant interaction with time interval (F5,80 = 3.18, P < 0.05), but not day (F4,64 = 2.45, P > 0.05). An interaction between time interval and day was also significant (F20,320 = 2.28, P < 0.01). A three–way interaction failed to achieve statistical significance (F20,320 = 0.76, P > 0.05). Because genotype had a significant interaction with time interval, this effect was further analyzed. First, we examined whether the time interval effect was significant in both WT and MAO-B KO mice. A two-way anova, including time interval and day, showed that time interval was significant for WT mice (F5,225 = 50.76, P < 0.005) and for MAO-B KO mice (F5,175 = 11.22, P < 0.005). Next, we determined which time interval contributed to the significant genotype effect. A two-way anova, including genotype and day, showed that WT and MAO-B KO mice differed at 25 min (F1,16 = 13.31, P < 0.008) and 30 min (F1,16 = 16.43, P < 0.0017), but not during the first 20 min. The data suggest that MAO-B KO mice retained a higher level of locomotion essentially during the last 10 min, as compared to WT mice.

image

Figure 1. Locomotor activity in WT and MAO-B KO mice. WT (n = 10) and MAO-B KO mice (n = 8) were tested in an inescapable open field for 30 min/day for 5 days. Data are expressed as average distance travelled at each 5-min bin per 30 min test period ± SEM.

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MAO-B KO and WT mice exhibit similar levels of oral nicotine intake

The cumulative amount of nicotine consumed was recorded every three days, and the corresponding data from Days 4, 7, 10, 13 and 16 are shown (Fig. 2a). We used a three-way anova, including genotype (WT vs. MAO-B KO), nicotine concentration (3.125–100 µg/ml) and day (Days 4–16, repeated measure). MAO-B KO and WT mice exhibited indistinguishable levels of nicotine intake (F1,70 = 0.58, P > 0.05). Both groups exhibited similarly higher levels of nicotine intake at higher concentrations of nicotine, as shown by a significant concentration effect (F5,70 = 26.23, P < 0.01). Both WT and MAO-B KO mice consumed up to approximately 15 mg/kg/3 days (i.e. 5 mg/kg/day). There was no daily fluctuation of nicotine intake (F4,280 = 0.44, P > 0.05). Genotype had no interaction with concentration (F5,70 = 0.23, P > 0.05) or with day (F4,280 = 2.03, P > 0.05). However, there was a significant interaction between concentration and day (F20,280 = 4.32, P < 0.01). A three–way interaction was also significant (F20,280 = 1.89, P < 0.05). These interactions are likely to reflect daily fluctuations in nicotine intake at some concentrations in both WT and MAO-B KO mice.

image

Figure 2. Nicotine consumption and preference/aversion in WT and MAO-B KO mice. (a) Voluntary nicotine intake. Data are expressed as average nicotine consumption (mg/kg/3 days ± SEM) against a log scale of nicotine concentrations (3.125, 6.25, 12.5, 25, 50 or 100 free base µg/ml). (b) Nicotine preference/aversion in WT and MAO-B KO mice. Each point represents the average ratio of nicotine intake to total fluid intake from the nicotine and water bottles during each three-day period (± SEM) against a log scale of nicotine concentrations. (WT, n = 6–8 and MAO-B KO, n = 6–9 per nicotine concentration).

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Nicotine preference/aversion ratios were analyzed using a three-way anova, including genotype (WT vs. MAO-B KO), concentration (0–100 µg/ml) and day (Days 4, 7, 10, 13 and 16, repeated measure) (Fig. 2b). The ratio was calculated by dividing the amount of nicotine intake by the total fluid intake, including water and nicotine solution, for each recording period. The ratios higher and lower than 0.5 indicate that mice preferred and avoided nicotine solution as compared to water, respectively. There was no significant difference in this ratio between WT and MAO-B KO mice (F1,84 = 1.88, P > 0.05). A statistically significant main concentration effect was found (F6,84 = 46.02, P < 0.01), reflecting an inverted-U curve with the highest levels of preference exhibited at concentrations of 6.25 and 12.5 µg/ml and aversion at 50 and 100 µg/ml. The main day effect also achieved statistical significance (F4,336 = 6.74, P < 0.01), suggesting that both WT and MAO-B KO mice showed similar daily fluctuations in nicotine preference/aversion. Genotype had a significant interaction with day (F4,336 = 3.82, P < 0.05), but not with concentration (F6,84 = 1.70, P > 0.05). The significant interaction between genotype and day is likely to reflect an isolated incidence of higher nicotine preference in WT than MAO-B KO mice at some concentrations on Day 10. However, there was no overall genotype effect and this isolated difference was not observed when nicotine intake was analyzed as doses (see Fig. 2a). An interaction between concentration and day was significant (F24, 336 = 3.82, P < 0.01). This suggests that WT and MAO-B KO mice exhibited equal daily fluctuations in nicotine preference/aversion at some concentrations. No three–way interaction was found (F24,336 = 1.35, P > 0.05).

WT and MAO-B KO mice did not exhibit significant differences in levels of nicotine intake or preference/aversion. We further analyzed the data to determine whether this result could have been confounded by differences between the total fluid intake or body weight of WT and MAO-B KO mice, as these parameters were used to calculate preference/aversion and consumed doses.

We first analyzed the total fluid intake of WT and MAO-B KO mice, using a three-way anova, including genotype (WT vs. MAO-B KO), concentration (0–100 µg/ml) and day (Days 4, 7, 10, 13 and 16, repeated measure) (see Table 1). The total fluid intake is the sum of the volume that mice drank from the water bottle and the nicotine bottle for each 3-day period; for the 0 µg/ml group, it was the sum of the volume that mice drank from the two water bottles. WT and MAO-B KO mice did not differ in their total fluid intake (F1,84 = 0.62, P > 0.05). However, there were significant main effects for concentration (F6,84 = 8.51, P < 0.01) and day (F4,336 = 28.29, P < 0.01). A significant interaction was found between genotype and day (F4,336 = 4.67, P < 0.01), but not between genotype and concentration (F6,84 = 0.93, P > 0.05). A significant interaction was found between concentration and day (F24,336 = 4.31, P < 0.01). A three-way interaction also reached significance (F24,336 = 1.74, P < 0.05). Because a three-way interaction was significant, the data were further analyzed for each concentration using two-way anovas, including genotype and day (see Table 1). WT and MAO-B KO mice did not differ at any concentration (see Genotype effect, Table 1). Significant daily fluctuations were found for 3.125, 6.25 and 50 µg/ml (see Day effect, Table 1), but there was no consistent tendency towards an increasing or decreasing trend. Furthermore, these daily fluctuations in fluid intake also occurred at the 0 µg/ml concentration, suggesting that they do not reflect the effect of nicotine.

Table 1.  Average total cumulative volumes of water (water and nicotine containing water in ml) consumed for each 3-day recording period
Concentration (µg/ml)GenotypeDay 4Day 7Day 10Day13Day16GenotypeDayInteraction
  1. SEM is shown in parentheses. WT, wild-type. KO, MAO-B knockout mice. NS, non-significant effect. The minimum level of significance accepted is adjusted by Bonferroni's correction.

0WT19.4 (1.0)13.8 (1.1)16.5 (1.5)14.5 (1.1)16.8 (0.7)NSP < 0.0014NS
 KO19.3 (0.7)15.8 (0.8)18.3 (1.0)14.6 (0.8)17 (0.9)   
3.125WT20.8 (1.4)16.8 (0.7)17.5 (0.7)15.7 (1.1)18.8 (0.9)NSP < 0.0014NS
 KO18.0 (0.8)14.5 (1.1)18.6 (0.8)15.5 (1.4)15.9 (1.0)   
6.25WT20.0 (1.3)17.6 (1.7)18.4 (1.0)19.6 (0.6)19.0 (0.5)NSP < 0.0014NS
 KO19.3 (0.8)17.4 (1.2)21.1 (0.8)17.4 (1.4)19.3 (0.8)   
12.5WT21.7 (1.3)19.4 (0.9)20.1 (1.2)20.1 (0.8)18.9 (0.7)NSNSNS
 KO18.5 (0.5)19.0 (0.7)19.7 (0.9)20.2 (1.0)20.0 (1.0)   
25WT20.0 (1.1)19.3 (0.6)19.3 (1.7)17.8 (0.5)19.5 (0.9)NSNSNS
 KO19.7 (0.3)20.7 (0.3)22.3 (1.2)21.0 (1.0)20.3 (0.7)   
50WT16.7 (1.1)16.0 (1.0)18.7 (1.2)15.7 (1.0)18.3 (1.5)NSP < 0.0014NS
 KO16.7 (0.7)14.3 (1.5)19.0 (1.0)14.8 (0.5)17.5 (0.7)   
100WT15.3 (0.8)16.4 (1.2)17.3 (1.3)16.2 (1.0)17.3 (0.2)NSNSNS
 KO16.8 (1.2)17.3 (1.2)17.7 (1.0)17.1 (1.2)16.5 (0.7)   

Next, we analyzed the body weight of WT and MAO-B KO mice using a three-way anova, including genotype (WT vs. MAO-B KO), concentration (0–100 µg/ml) and day (Days 4, 7, 10, 13 and 16, repeated measure, see Table 2). No main effect was found for genotype (F1,84 = 3.65, P > 0.05) or concentration (F6,84 = 1.08, P > 0.05). The main effect was significant only for day (F4,336 = 31.26, P < 0.01). There was a significant interaction between genotype and day (F4,336 = 5.64, P < 0.01), but not between genotype and concentration (F6,84 = 1.59, P > 0.05). Interaction was significant between concentration and day (F24,336 = 2.52, P < 0.01). There was no significant three-way interaction effect (F24, 336 = 0.71, P > 0.05). Because studies have shown that high doses of nicotine retard weight gain over days (Grunberg et al. 1987; Winders & Grunberg 1990) and an interaction was significant between concentration and day, we wished to determine which concentration contributed to the day effect. We analyzed the data for each concentration using two-way ANOVAs, including genotype and day (see Table 2 for results of this analysis). There was no difference in body weight between WT and MAO-B KO mice at any nicotine concentration tested (see Genotype effect, Table 2). A daily increase in body weight was evident up to 25 µg/ml nicotine for WT and MAO-B KO (see Day effect, Table 2). However, at the two highest concentrations (50 and 100 µg/ml), neither WT nor MAO-B KO mice significantly gained body weight over the duration of the study (see Day effect).

Table 2.  Average body weight (g) at the end of each 3-day recording period
Concentration (µg/ml)GenotypeDay 4Day 7Day 10Day13Day16GenotypeDayInteraction
  1. SEM is shown in parentheses. WT, wild-type. KO, MAO-B knockout mice. NS, non-significant difference effect. The minimum level of significance accepted is adjusted by Bonferroni's correction.

0WT22.8 (0.97)23.8 (0.80)24.0 (0.92)24.1 (0.92)24.3 (0.86)NSP < 0.007NS
 KO25.3 (0.72)25.6 (0.76)26.1 (0.65)26.3 (0.67)26.7 (0.56)   
3.125WT23.7 (1.53)23.8 (1.49)23.9 (1.37)23.8 (1.48)23.9 (1.37)NSP < 0.007NS
 KO24.0 (0.84)24.6 (0.97)24.7 (0.95)24.9 (0.89)24.9 (0.73)   
6.25WT22.0 (0.91)23.0 (0.84)23.6 (0.83)23.2 (0.91)23.3 (0.83)NSP < 0.0014NS
 KO24.0 (0.83)24.4 (0.83)24.6 (0.88)24.7 (0.81)25.2 (0.91)   
12.5WT23.3 (0.30)23.3 (0.35)23.9 (0.34)23.6 (0.31)24.2 (0.27)NSP < 0.0014NS
 KO22.3 (1.71)22.3 (1.58)23.0 (1.69)22.9 (1.72)23.9 (1.55)   
25WT22.4 (0.40)22 (0.40)22.3 (0.25)22.3 (0.25)22.7 (0.22)NSP < 0.0014NS
 KO24.2 (0.71)23.7 (0.87)23.9 (0.93)24.1 (0.88)24.7 (0.84)   
50WT23.1 (0.61)22.9 (0.34)22.9 (0.35)23.3 (0.38)23.2 (0.30)NSNSNS
 KO24.8 (1.01)24.9 (0.94)25.1 (0.90)25.1 (1.05)25.0 (1.07)   
100WT22.4 (1.08)22.7 (0.87)23.0 (0.82)23.1 (0.82)22.3 (1.31)NSNSNS
 KO23.7 (0.56)23.9 (0.61)24.1 (0.61)24.5 (0.66)24.4 (0.64)   

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The present study shows that deletion of the MAO-B gene is associated with an attenuated reduction of locomotor response during prolonged exposure to an inescapable open field, but it does not affect nicotine intake; the latter effect did not reflect any confounding abnormality in the total fluid intake or body weight of MAO-B KO mice. A large body of evidence suggests that the locomotor response measured by this method is a reliable predictor of the reinforcing and rewarding effects of nicotine and other abused drugs in rodents (see Introduction for references). Our findings provide evidence that the MAO-B gene does not concomitantly contribute to both the regulation of locomotor activity and voluntary nicotine intake.

MAO-B and behavioral response in an inescapable open field

WT mice showed a gradual decline in motor activity when repeatedly exposed to the test environment over a period of 5 days and when continuously exposed to this environment for 30 min within each session. When animals are placed in an inescapable open field, they exhibit locomotor activity. This behavior is thought to represent their responsiveness to novelty (Bardo et al. 1996; Crawley 2000; Dellu et al. 1996; Laviola et al. 1999). Our inescapable open field was relatively small (26 cm × 26 cm × 38.5 cm) and placed under dim illumination (approximately 97 lux), conditions known to reduce anxiety and to maximize the locomotor response to a novel environment in rodents (Crawley 2000). Day 1 could be considered to be the only day that mice perceived the environment to be highly novel. However, WT mice continued to exhibit decreases in locomotor activity within sessions and across sessions over a period of 5 days. If Day 1 were the only day in which mice perceived the environment to be novel, no further decline in locomotor activity after Day 1 would be expected. Because this was not the case, it is likely that a certain degree of novelty was retained over a period of 5 days and that the gradual decline in locomotor activity within and across sessions reflects their habituation to a novel environment (see Crawley 2000; File 2001).

In contrast, our data suggest that MAO-B KO mice exhibited higher levels of locomotor activity after prolonged exposure to the test environment, as compared to WT mice. Grimsby et al. (1997) demonstrated that MAO-B KO mice and WT mice did not exhibit differences in their locomotor activity in an inescapable open field when animals were tested for 10 min on a single day. Our finding is consistent with this observation, in that the locomotor activity of MAO-B KO and WT mice did not differ during the first 20 min. The present study further extends their finding and suggests that MAO-B KO mice exhibit consistent reductions in within-session habituation toward the end of each session.

Alternatively, this phenotype could be interpreted as suggesting that MAO-B KO mice simply showed a higher level of baseline motor activity compared to WT mice. However, this interpretation is inconsistent with the fact that both groups initially exhibited indistinguishable levels of motor activity during the first 20 min of each day. Mill et al. (2002) showed that mice that show high and low baseline levels of motor activity in their home cage are indistinguishable in the first 2 min in an inescapable open field, but become differentiated after the third minute within a 5-min session of a single testing day. If general baseline motor activity was increased in MAO-B KO mice as compared to WT mice, this would be expected to become apparent in the first 5 min on Day 1. No such difference was found between WT and MAO-B KO mice on Day 1 (see Fig. 1, Day 1). Moreover, if there was absolutely no deficit in habituation in MAO-B KO mice and sustained high levels of motor activity in MAO-B KO mice simply reflected high levels of baseline motor activity, one would expect the decline in locomotor activity within a day to be parallel between WT and MAO-B KO mice over days. This was not the case, as there was a significant overall interaction between genotype and time interval (see Fig. 1). Although the present study does not entirely exclude the possibility that the sustained locomotor response of MAO-B KO mice during the last 10 min of each session simply reflects heightened baseline motor activity, it is likely that MAO-B KO mice have at least an additional impairment in within-session habituation in locomotor activity.

It is interesting to note that WT and MAO-B KO mice differed during the last 10 min of each session. One possible interpretation of this finding is that MAO-B KO mice show a generalized memory deficit, as habituation is considered to be a form of memory (File 2001). This interpretation is not plausible, because the MAO-B KO mice performed normally in the Morris water maze (Holschneider et al. 1999). However, it should be noted that there is conflicting evidence regarding the role of MAO-B in memory. Barber et al. (1993) demonstrated that long-term inhibition of MAO activity in mice impairs spatial learning. A memory deficit specific to within-session habituation might underlie the sustained locomotor activity in MAO-B KO mice.

The deletion of the MAO-B gene does not alter MAO-A activity or the levels of its substrates (i.e. dopamine, serotonin and norepinephrine) and their metabolites (Grimsby et al. 1997). Thus, there is no compensatory up-regulation of MAO-A in the absence of MAO-B in mice. Rather, deletion of MAO-B selectively increases phenylethylamine levels in mice (Grimsby et al. 1997). Phenylethylamine is enriched within the nucleus accumbens (see Paterson et al. 1990). Because individual differences in behavioral responsiveness in an inescapable open field are partly determined by neurochemical differences in the nucleus accumbens (Bradberry et al. 1991; Chefer et al. 2003; Hooks & Kalivas 1995; Hooks et al. 1991, 1992; Jones et al. 1996; Marinelli & Piazza 2002; Marinelli & White 2000; Piazza et al. 1991b; Rouge-Pont et al. 1993, 1998; Zhuang et al. 2001), an increased level of phenylethylamine within the nucleus accumbens might have prevented MAO-B KO mice from habituating to an inescapable open field.

The mechanism by which deletion of MAO-B alters rodent behavior needs to be considered. MAO-B deletion could have affected locomotor habituation either directly or indirectly. It could be that MAO-B directly mediates the ability of animals to habituate to an inescapable open field. Alternatively, deletion of MAO-B might have affected maternal behavior, which could indirectly affect the habituation behavior exhibited by MAO-B KO offspring. Because our mice were from a congenic line, we paired male and female MAO-B KO mice to generate MAO-B KO offspring and male and female WT mice to generate WT offspring. If deletion of MAO-B caused abnormalities in maternal behavior, MAO-B KO, but not WT offspring would be affected. In fact, there is evidence suggesting that neonatal stress has long-lasting effects on the responsiveness of dopamine and serotonin systems in the brain (Papaioannou et al. 2002). However, several findings are inconsistent with this possibility. If abnormal maternal behavior of MAO-B KO mothers affected their MAO-B KO offspring, there might be some global impact on the behavior of MAO-B KO offspring. WT and MAO-B KO mice did not differ in nicotine intake (as we show here) or in spatial learning (Holschneider et al. 1999). Nor did WT and MAO-B KO mice differ in their initial locomotor activity during the first 20 min of each day. Although more work is needed to determine whether deletion of MAO-B affects locomotor habituation via abnormal maternal behavior, it is highly unlikely that abnormal maternal behavior could so selectively affect habituation of locomotor activity of the offspring in an inescapable open field.

MAO-B and nicotine intake

Both WT and MAO-B KO mice consumed nicotine in a concentration-dependent manner (see Fig. 2a). This pattern is in close agreement with other studies demonstrating that oral nicotine intake occurs in a concentration-dependent manner (Adriani et al. 2002; Flynn et al. 1989; Klein et al. 2003; Meliska et al. 1995a, b; Robinson et al. 1996). The concentrations used in our nicotine intake experiment are comparable with those used in other studies (Flynn et al. 1989; Klein et al. 2003; Le Houezec et al. 1989; Robinson et al. 1996). Our findings also show that mice exhibit preference for nicotine at low concentrations and aversion at high concentrations (see Fig. 2b). The concentration-dependent dual actions of nicotine are also observed in the conditioned place preference method, using a different route of administration (Fudala & Iwamoto 1986; Horan et al. 1997; Jorenby et al. 1990; Martin & Itzhak 2000; Risinger & Oakes 1995).

In the oral intake method, mice had a choice of drinking from either a bottle that contained water or a bottle that contained a nicotine solution. Although the mice clearly preferred water to nicotine at higher concentrations (50 and 100 µg/ml, see Fig. 2b), they nevertheless continued to consume a limited amount of nicotine-containing water and never ceased drinking nicotine at high concentrations, despite their relative aversion for it, as compared to water. This is reflected by the somewhat flattened increase in nicotine intake at the two highest concentrations (see Fig. 2a); even if a mouse consumed half the amount of nicotine-containing water at a concentration that was twice as high, the overall nicotine dose consumed would be the same. Moreover, the preference ratio for nicotine never reached zero at these concentrations (see Fig. 2b). Taken together, this could suggest that the net effects of nicotine are more rewarding than aversive at low concentrations, and vice versa at high concentrations. At a minimum, these findings suggest that fluid intake from the nicotine-containing bottle was determined by the concentration of nicotine, but not by a pre-existing position preference or other factors.

At nicotine concentrations of up to 25 µg/ml, WT and MAO-B KO mice gained body weight equally over the 16-day testing period. However, both WT and MAO-B KO mice that received the two highest concentrations of nicotine (50 and 100 µg/ml) failed to gain body weight over the 16-day period (Table 2). At these high concentrations, mice drank approximately 10–15 mg/kg/3 days (i.e. 3–5 mg/kg/day). It has been reported that high doses of nicotine (6 and 12 mg/kg/day via osmotic minipump) delay body weight gain in rats (Grunberg et al. 1987; Winders & Grunberg 1990). Taken together with the concentration-dependent alterations in nicotine intake, it is likely that nicotine exerts pharmacological effects in our oral intake method. Furthermore, the effect of nicotine on body weight strengthens the argument that orally self-administered nicotine achieves bioavailability.

Our data show that WT and MAO-B KO mice did not differ in oral nicotine intake over a wide range of concentrations. This finding suggests that variations in MAO-B activity alone are not sufficient to alter susceptibility to voluntary nicotine intake. Our experimental procedure was designed to tip the balance in favor of nicotine consumption (see Materials and methods for details), and it is unlikely that the procedure was not sufficiently sensitive to detect a subtle difference between WT and MAO-B KO mice. One commonly used drinking method alternates the position of a drug-containing bottle and a water bottle, and data are usually collapsed across days. As discussed in Materials and methods, this switched procedure would introduce high levels of variance into data. More critically, drinking behavior in the switched procedure depends on the ability of the animal to discriminate stimuli and to reverse an established behavior, as well as on the reinforcing effects of a drug. This is problematic, as these and other KO mice show abnormalities in reversing an established behavior (Buhot et al. 2003; Heyser et al. 2000; Holschneider et al. 1999).

It is important to note that there is a species difference in the specificity of the interactions of MAOs with the neurotransmitter dopamine. In humans, MAO-A preferentially oxidizes serotonin and norepinephrine, whereas MAO-B oxidizes dopamine, phenylethylamine and benzylamine (Lan et al. 1989; Shih et al. 1999). However, in rodents, MAO-A oxidizes serotonin, norepinephrine and dopamine, while MAO-B oxidizes phenylethylamine and benzylamine (Cases et al. 1995; Grimsby et al. 1997; Johnston 1968; Neff & Yang 1974). Despite this species difference, our data are consistent with recent findings demonstrating that MAO-B polymorphisms do not affect smoking risk (Hernan et al. 2002; Tan et al. 2003).

Genetic basis for nicotine addiction and comorbid behavioral traits

It has been suggested that genetic influences on smoking are mediated partly by individual differences in comorbid personality traits, as well as individual differences in the reinforcing effects of nicotine (Lerman & Niaura 2002). Altered responsiveness to novelty is thought to be associated with a heightened susceptibility to smoking in humans (Heath et al. 1995; Kopstein et al. 2001; Wills et al. 1994; Zuckerman & Kuhlman 2000). In humans, inherent MAO-B activity levels have been found to be correlated with novelty- and sensation-seeking (Zuckerman & Kuhlman 2000) as well as smoking (Oreland et al. 2002).

The present study raises two critical issues regarding the relationship between nicotine addiction and comorbid traits. First, exactly what behavioral trait is associated with individual differences in the addictive impact of drugs in animals? While the human literature uses a wide range of terms, including ‘novelty seeking’ and ‘sensation seeking’, it remains unclear exactly what aspect of behavioral response to novel environments is correlated with smoking risks. There are a number of methods that purportedly measure an animal's response to novelty. They include locomotor activity in an inescapable open field (Grimm & See 1997; Klebaur et al. 2001; Nadal et al. 2002; Piazza et al. 1989; Pierre & Vezina 1997; Rubinstein et al. 1997; Suto et al. 2001), preference for a novel compartment (Bardo et al. 1996; Misslin & Cigrang 1986; Misslin & Ropartz 1981a,b), exploration of a novel object (Dulawa et al. 1999; Klebaur et al. 2001; Misslin & Ropartz 1981a; Nicholls et al. 1992) and the novelty Y-maze (Dellu et al. 1996). Klebaur et al. (2001) demonstrated that locomotor activity in an inescapable open field is the only behavior that reliably predicts individual variations in the reinforcing effects of amphetamine; neither exploration of a novel object nor novelty place preference predicts the degree of amphetamine self- administration.

What is then uniquely represented in the behavior seen in an inescapable open field? Belzung (1999) argued that locomotor activity measured in an inescapable open field is not a measure of exploration, but of activity and anxiety. Moreover, Misslin & Cigrang (1986) reported that mice showed significant elevations in plasma corticosterone levels when they were forced into a novel environment but mice showed little change in plasma corticosterone levels when they had a free choice between a novel environment and a familiar environment. Their study elegantly demonstrated that novelty per se did not automatically induce anxiety and fear, but mice did exhibit anxiety and fear toward a novel environment when they were forced into it. Consistent with this, Dellu et al. (1996) demonstrated that individual differences in the degree of corticosterone increases in a forced novel environment predict the reinforcing effects of abused drugs in rats. Finally, we previously reported that MAO-B KO mice showed a blunted habituation in the forced swim test, a form of stress test, upon repeated testing (Grimsby et al. 1997). However, at the onset of testing, MAO-B KO mice exhibited a similar level of swimming as WT mice. The locomotor activity seen in the present study is similar to this phenotype in terms of the initial response as well as habituation. Because it is possible that locomotor activity seen at the onset of each session reflects a non-specific behavioral arousal related to the stress inherent to the inescapable open field, MAO-B KO mice may have a blunted habituation response to the stress (see also Klebaur et al. 2001; Piazza et al. 1991a).

Although these studies clearly indicate that stress factors could be involved in the locomotor response in a novel inescapable open field, they do not entirely rule out the possibility that such locomotor activity also involves behavioral responses to novelty other than stress-associated anxiety and fear (see Prut & Belzung 2003). More work is needed to identify what behavioral factor is uniquely associated with nicotine addiction in humans and rodents.

The second issue raised by this study is that the role of genes in nicotine addiction and comorbid traits is likely to be complex. It was unclear whether altered MAO-B activity causally influences both locomotor response in an inescapable open field and voluntary nicotine intake. The data from this study now rule out the MAO-B gene as a common denominator for both of these behaviors. Because altered locomotor response in an inescapable open field does not necessarily affect nicotine intake or preference/aversion, genetic modulation of smoking risks could occur independently of altered comorbid behavioral traits. It is conceivable that variations in the activity of other genes influence both smoking and responsiveness to novelty, or only smoking. Given the highly polygenic nature of smoking and novelty responsiveness, this is a plausible hypothesis that warrants further investigation. In fact, fosB knockout mice initially show higher locomotor activity in an inescapable open field and exhibit a heightened behavioral response to the motor activating and rewarding effects of cocaine (Hiroi et al. 1997). Taken together, these findings represent an important step towards understanding the genetic mechanisms underlying the complex comorbid traits of addiction.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

This work was supported by Program in Human Genetics/Howard Hughes Medical Institute and funds from Dr T. Byram Karasu, Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine to NH, and NIMH R37-MH39085 (Merit Award) and Welin professorship to J.C.S. We thank the late T. Klein for support.