MELATONIN RECEPTOR KNOCKOUT MICE HAVE AN INCREASED PHYSIOLOGICAL REACTION TO NICOTINE AND INCREASED VOLUNTARY ORAL NICOTINE CONSUMPTION.

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1 MELATONIN RECEPTOR KNOCKOUT MICE HAVE AN INCREASED PHYSIOLOGICAL REACTION TO NICOTINE AND INCREASED VOLUNTARY ORAL NICOTINE CONSUMPTION. by Sheila Irene Bridget Maier A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Bachelor of Arts/Master of Science Department of Integrative Physiology 2008

2 UMI Number: UMI Microform Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI

3 This thesis entitled: Melatonin receptor knockout mice have an increased physiological reaction to nicotine and increased voluntary oral nicotine consumption written by Sheila Irene Bridget Maier has been approved for the Department of Integrative Physiology Jerry Stitzel Marissa Ehringer Date: April 8, 2008 The final copy of this thesis has been examined by the signatories, and we Find that both the content and the form meet acceptable presentation standards Of scholarly work in the above mentioned discipline.

4 Maier, Sheila Irene Bridget (B.A., Department of Integrative Physiology) Melatonin receptor knockout mice have an increased physiological reaction to nicotine and increased voluntary oral nicotine consumption. Thesis directed by Assistant Professor Jerry Stitzel, Ph.D Abstract Melatonin is a ubiquitous hormone common to nearly all major taxonomic groups. It has long been known to function in regulating circadian rhythms and sleep, but recent research has shown that melatonin has a more complex role. Melatonin has been implicated in modifying an organism s response to drugs of abuse, such as cocaine and nicotine. Specifically, melatonin appears to attenuate the physiological response to these drugs. We used melatonin receptor knockout mice to characterize the role of melatonin receptors in regulating nicotine response over the 24-h circadian cycle. We used three measures of nicotine sensitivity; nicotine-induced hypothermia, nicotineinduced locomotor depression and voluntary oral nicotine consumption. We tested four genotypes, C3H/IBG mice, melatonin 1a knockout mice and their wild-type littermates, as well as melatonin 1a1b receptor knockout mice on a C3H/HeN background. Normal C3H mice have a significantly greater nicotine-induced hypothermia and locomotor depression in the light phase than in the dark phase of the circadian cycle. Melatonin 1a knockouts and their wild-type littermates both had attenuated response to nicotine, but no significant variation between the light and dark phases. Melatonin 1a knockouts and the wild-types also self-administer significantly less nicotine. iii

5 Melatonin 1a1b knockout mice have increased sensitivity to nicotine, as measured by nicotine-induced hypothermia and locomotor depression, and do not have a consistent variation between the light and dark phases. Melatonin 1a1b knockouts also have increased voluntary oral nicotine consumption. Male melatonin 1a knockout mice, male wild-types, and male melatonin 1a1b receptor knockout mice also had increases sensitivity to nicotine-induced hypothermia and nicotine-induced locomotor depression compared to their female counterparts. We conclude that melatonin receptors play a role in modulating the physiological response to nicotine, especially in the dark phase of the circadian cycle. iv

6 Acknowledgements First of all, I would like to thank my parents, Karen and John. They have always supported and guided me, even when it meant another year of college. Without them I would have never made it this far. I also want to thank all of friends, mostly for believing in me even when I didn t. You know who you are. I d also like to thank my fellow grad students for their encouragement and commiseration. I would like to thank Dr. Jerry Stitzel for giving me this project and letting me run with it. His comments and suggestions were invaluable. Thanks also go to Dr. Sharon Mexal, who was an advisor and mentor during my undergraduate years. Jennifer Wilking and Eric Crouch provided endless help with designing and running these protocols, as well as assisting with the final data analysis. Lastly, I d like to thank Amy Hua, Andra Wilkinson, Sonia Ciesnik, and Marisa Maroslek for their help in data collection.

7 Table of Contents 1. Introduction.. pg Methods and materials a. Animals pg. 5 b. Locomotion and Body Temperature Protocol.. pg. 5 c. Two-Bottle Preference Protocol.. pg Results a. Locomotion & Body Temperature:Light v. Dark phase (1.0 mg/kg nicotine)... pg.8 b. Locomotion & Body Temperature: 1.0 mg/kg nicotine i hours relative clock time... pg. 14 ii hours relative clock time... pg. 20 c. Locomotion & Body Temperature: 0.5 mg/kg nicotine...pg. 21 d. Locomotion & Body Temperature: 1.5 mg/kg nicotine. pg. 24 e. Two-Bottle Preference... pg. 27 i. Nicotine Preference by Sex pg Discussion a. Locomotion & Body Temperature:Light v. Dark phase(1.0 mg/kg nicotine).... pg.33 b. Locomotion & Body Temperature: 1.0 mg/kg nicotine i. 1.0 mg/kg nicotine: 0400 h relative clock time... pg. 34 ii. 1.0 mg/kg nicotine: 1600 h relative clock time... pg. 35 c. Locomotion & Body Temperature: 0.5 mg/kg nicotine (0400 h & 1600 h relative clock time)....pg. 36 d. Locomotion & Body Temperature: 1.5 mg/kg nicotine (0400 h & 1600 h relative clock time)..... pg. 38 e. Summary of Locomotion and Body Temperature.....pg. 38 f. Two-Bottle Preference... pg. 39 g. Further Research.... pg Conclusion..... pg References..pg. 44 vi

8 List of Tables 1. ANOVA of Nicotine Consumption. pg. 30 List of figures 1. Plasma Melatonin Levels in C3H mice... pg Body Temperature: Light v. Dark phase.... pg Male Body Temperature: Light v. Dark phase pg Female Body Temperature: Light v. Dark phase pg Locomotion: Light v. Dark phase pg Male Locomotion: Light v. Dark phase. pg Female Locomotion: Light v. Dark phase. pg Body temperature: 1.0 mg/kg nicotine... pg Locomotion: 1.0 mg/kg nicotine pg Male Body temperature: 1.0 mg/kg nicotine..... pg Female Body temperature: 1.0 mg/kg nicotine pg Male Locomotion: 1.0 mg/kg nicotine... pg Female Locomotion: 1.0 mg/kg nicotine pg Body temperature: 0.5 mg/kg nicotine..... pg Locomotion: 0.5 mg/kg nicotine....pg Body temperature: 1.5 mg/kg nicotine... pg Locomotion: 1.5 mg/kg nicotine.... pg Nicotine Consumption: % of total consumption volume... pg Nicotine Consumption (mg/kg).... pg. 28 vii

9 Introduction Melatonin is molecule with complex and wide-ranging functions in the body. In most vertebrates, including mice and humans, it is secreted from the pineal gland in response to circadian cues. Melatonin secretion is suppressed by bright light and upregulated during the dark phase of an organism s 24-hour cycle, regardless of whether or not the organism is nocturnal. Thus, melatonin is an entrainment cue, synchronizing an organism s intrinsic circadian cycle with the external circadian cycle. In C3H mice, peak melatonin levels occur around 0400 hours relative clock time and the lowest levels occur around hours (Figure 1). There are two melatonin receptor subtypes in most vertebrates, including mice and humans, called Melatonin 1a, or MT 1, and Melatonin 1b, also called MT 2. A third subtype, Melatonin 1c, has been characterized in birds and amphibians, but not in mammals (Reppert et al., 1996). Both receptors are expressed in the suprachiasmatic nucleus and in peripheral organs, and both function in regulating circadian biology, but through slightly different pathways. Among other things, melatonin 1a suppresses neuronal firing, while melatonin 1b induces phase shifts (Pandi-Perumal et al., 2006). Melatonin s primary role appears to be in regulating the body s internal clock, but it also appears to play a role in major depression and mood disorders, protecting the GI tract, regulating blood pressure and enhancing immune function (Pandi-Perumal et al., 2006). The literature on sex differences and melatonin is contradictory. Weil et al. (2005) found female mice had increased locomotion compared to males among wild-type melatonin knockout mice, but not among melatonin1a knockout mice. In humans, Monteleone et al. (1995) found a significant sex difference in melatonin suppression in response to bright light, while Nathan et al. (2000) found no significant effect. These studies vary in methodology and both examine the alteration of melatonin levels in response to bright light, rather than normal nighttime levels. 1

10 This contradictory evidence suggests that if there is a sex difference in melatonin expression in mice and humans, the difference is small and not easily elucidated. Recent studies have also shown that melatonin may play a role in regulating response to drugs of abuse. Sensitivity to the rewarding effects of cocaine has been shown to vary with the circadian cycle. Mice lacking a functional Clock gene, which functions in circadian entrainment, showed an increased sensitivity to the rewarding effects of cocaine and increased activity in dopaminergic neurons of the VTA (Nestler et al., 2005). Akhisaroglu et al. (2004) showed that melatonin-deficient AKR mice became sensitized to the rewarding effects of cocaine when cocaine was injected at night, while melatonin-proficient CBA mice did not. Kurtuncu et al. (2004) found that C3H mice showed place preference in response to cocaine only in the light phase, while pinealectomized C3H mice showed a preference in the light and dark phase. These studies suggest that the time of day and melatonin levels influence whether mice develop an affinity for cocaine. Melatonin has also been shown to modulate the effects of nicotine. Schiller et al. (2003) found that an acute dose of melatonin inhibited nicotine-stimulated dopamine release from PC12 cells, a rat adrenal medullary tumor cell line. Melatonin may also directly affect nicotinic acetylcholine receptors. Zago and Markus (1999) found that rat vas deferens cells, which express high-affinity nicotine binding sites in the light phase, begin to express low-affinity binding sites in the dark phase, and that this effect could be triggered in vitro with melatonin. An examination of the literature shows that strains of melatonin-proficient mice may be less sensitive to nicotine than mice that are melatonin-deficient. BALB/c and C57BL/6 produce melatonin, but the levels are usually at or below the detection threshold, while CBA and C3H mice produce significant amounts of melatonin, as well as having a defined circadian rhythm 2

11 (Viven-Roels et al., 1998). Collins et al. (1988) found that BALB and C57BL/6 mice require less nicotine than CBA and C3H mice to show a similar decrease in locomotion and body temperature. In humans, there is evidence that sensitivity to nicotine may vary over the 24-hour circadian cycle. A rare type of seizure, called autosomal dominant nocturnal frontal lobe epilepsy, has been linked with nicotinic receptor mutations, specifically in the α4 and β2 receptor subunits. The fact that this type of epilepsy occurs only at night suggests that nicotinic receptors are somehow altered at night (Combi et al, 2004). Drive to consume nicotine or the beneficial side-effects of smoking may also be regulated over the circadian cycle. Even heavy smokers (>15 cigarettes a day) will cease smoking for several hours at night while they sleep (Riedel et al, 2004). Research has also indicated that melatonin supplementation may alleviate some of the negative side-effects of smoking cessation, such as mood disorders (Zhdanova and Piotrovskaya, 2000). However, there has been little research to date on the specific influence of the circadian cycle on the physiological reaction to nicotine. In other words, what is the impact of the time of day on an organism s response to nicotine? In this series of experiments, we attempted to characterize not only the impact of time of day on the physiological reaction to nicotine in a mouse animal model, but also melatonin and melatonin receptors role in that response. We hypothesize that melatonin-proficient C3H mice will show a distinct variation in their response to nicotine between the light and dark phases of the circadian cycle. We also hypothesize that this variation is due to melatonin and mediated by the melatonin receptor, so that melatonin-receptor knockout mice will not show a variation in their response to nicotine between the light and dark phases. 3

12 Plasma melatonin levels relative to C3H ZT hr Plasma melatonin levels in C3H mice Zeitgeber Hour Figure 1: Plasma melatonin levels relative to ZT 1 in C3H mice. ZT 1 corresponds with 0700 relative clock time. Peak melatonin levels occur at about ZT 21 (relative clock hours: 0400) and lowest levels are at ZT 11 (relative clock hour: 1800). 4

13 Methods Animals The animals used in this experiment were C3H mice from the Institute for Behavioral Genetics (Boulder, CO), and melatonin receptor knockout mice on a C3H/HeN background, obtained from Dr. David Weaver (University of Massachusetts, Amherst, MA). Single knockout mice (sko) were knocked out for the melatonin 1a receptor. Wild-type mice (WT) were the wild-type littermates of the single knockout mice. Double knockout mice (dko) were knocked out for both melatonin 1a and 1b receptors; there were no wild-type littermates for the double knockout mice. We were unable to breed significant numbers of melatonin 1b receptor knockouts or their wild-type littermates, so these mice were excluded from the final analysis. The melatonin receptor knockout genotypes still produce melatonin normally. Animals were bred in the IBG animal facility, and moved to the adjacent testing facility colony room as adult mice. Mice were allowed to acclimate in the colony room for two weeks before testing began. Mice were separated by sex and housed with their littermates. Singly housed mice were provided with additional cotton bedding. The colony rooms were kept at 20 C (±1 ) and light levels were 300 lux (International Light photometer, Model IL1400A). All mice were given free access to food and water. These conditions and protocols were approved by the University of Colorado Institutional Animal Care and Use Committee. Body Temperature and Locomotion Protocol Previous testing indicated that melatonin levels were highest at 0400 h and lowest at 1700 h (Figure 1). We chose 0400 h and 1600 h as our two time points to gather data on change in behavior due to nicotine. We chose 0400 h since it was near the peak of plasma melatonin levels and 1600 h because it is exactly 12 hours from 0400 hours, even though according to our data, 1600 h is not the absolute nadir of plasma melatonin levels. 5

14 We used two measures to quantify the effect of nicotine in the mice, locomotion and body temperature. Locomotion was defined as the total number of rears and crosses in a 180-sec period. Locomotor activity was recorded with a symmetrical Y-maze. The Y-maze was constructed of red plastic; all three arms were 26 cm long, 6.1 cm wide and 10.2 cm high. Infrared beams for monitoring crosses were located in the middle of each arm and at the central exit. The beams for measuring rears were set 6.4 cm above the floor in each arm. Each rear and cross was counted as 1 locomotor unit. Body temperature was measured with a rectal thermometer (Bailey Instruments). First, baseline data were gathered. Mice were weighed and then injected with saline (injection volume =.1 ml/g, intraperitoneal). After 3 minutes, the mice were placed in a Y-maze and their locomotion recorded. Mice were removed from the maze and placed in separate cages. Body temperature was recorded 7 minutes after the mouse was removed from the Y-maze, approximately 16 minutes after the nicotine injection. After every injection trial, the mice were allowed to recover for at least 48 hours. After 48 hours, the mice were injected with a dose of nicotine of 0.5, 1.0, or 1.5 mg/kg and an injection volume of.1 ml/g. The locomotion and body temperature was then compared to the saline control. Saline control trials were repeated as necessary so that the baseline saline trial and nicotine trial were always between 48 and 216 hours apart (2-9 days). All mice were subjected to the 1.0 mg/kg injection protocol, but of the protocol length, some mice died of natural causes before they could be tested at the 0.5 mg/kg or 1.5 mg/kg time point. Mice of the C3H genotype were only tested at the 1.0 mg/kg time point. In order to measure locomotor activity and body temperature at the 0400 h time point, mice were housed in a reverse light-cycle colony room for 14 days to acclimate them to the 6

15 reverse light-cycle, then tested with the above procedure under dim red light. Light levels in the testing room were 300 lux for the 1600 hours condition and 8 lux for the 0400 hours condition. Two-Bottle Preference Protocol Mice were singly housed and given free access to food and water, as well as cotton bedding. Mice had access to two bottles of water, one containing tap water and one containing nicotine dissolved in tap water. Fluid consumption was measured every day and the bottles refilled as necessary. The bottles were rotated everyday to control for place preference, since animals tend to show a slight preference for the bottle closest to their food (Wilking, unpublished data). Total consumption of nicotine solution and water was measured over a period of four days, then the nicotine dosage was increased, starting with 25 μl nicotine per milliliter water, followed by 50 μl/ml and 100 μl/ml. To control for fluid lost to evaporation and while rotating the bottle, we also measured water and nicotine volume from an empty control cage. The nicotine and water volume lost from the control cage was subtracted from the respective consumption volumes for each mouse before data analysis. Statistical analysis was performed SPSS 15 for Windows (V , 2006) and graphs were constructed with SigmaPlot 2004 for Windows (v 9.01). 7

16 Results Locomotion & Body Temperature: Light v. Dark phase (1.0 mg/kg nicotine) First, we compared the change in body temperature and locomotion in the light and in the dark. Student s t-test was performed on the data collected after the mice were injected with 1.0 mg/kg nicotine. Previous research and experience within our lab had indicated that this dose appears to be optimum for eliciting differences without creating a ceiling effect (Marks, 1989). At this dose we had 30 C3H mice (female=16), 21 wild type mice (female=10), 19 single knockout mice (female=10) and 22 double knockout mice (female=9). For C3H mice, the mean decrease in body temperature (Figure 2) was much greater in the light than in the dark (X dark=-0.63 C±.10, X light=-2.42 C±.33, p=0.000). The mean decrease in locomotion (Figure 5) also varied significantly (X dark=-12.79±13.13, X light = ±13.97, p=0.000). For female C3H mice, there was a significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-0.52 C±.16, X light=-2.55 C±.57, p=0.004) (Figure 4) and the mean decrease in locomotion (X dark=-1.83±21.71, X light = ±19.70, p=0.001) (Figure 7). For male C3H mice, there was also a significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-0.75 C±.13, X light=-2.28 C±.35, p=0.001) (Figure 3) and the mean decrease in locomotion (X dark= ±15.11, X light = ±17.72, p=0.001) (Figure 6). The mean decrease in body temperature (Figure 2) for wild-type mice did not vary significantly between light and dark (X dark =-0.44 C±.09, X light =-0.75 C±.26, p=0.20). Their mean decrease in locomotion (Figure 5) also did not vary significantly (X dark =-5.25±8.31, X light =-20.75±10.68, p=0.242). For female wild-type mice, there was no significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-0.32 C±.10, X light=-0.51 C±.31, p=0.567) (Figure 4) and or the mean decrease in locomotion 8

17 (X dark=9.66±11.53, X light = -7.33±14.24, p=0.341) (Figure 7). For male wild-type mice, there was also no significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-0.60 C±.16, X light=-1.05 C±.44, p=0.236) (Figure 3) and the mean decrease in locomotion (X dark=-24.42±7.61, X light = ±14.72, p=0.542) (Figure 7). The single knockout mice s mean decrease in body temperature (Figure 2) did not vary significantly between light and dark (X dark =-0.71 C±.17, X light =-0.52 C±.16, p=0.389). Their mean decrease in locomotion (Figure 5) also did not vary significantly (X dark =-15.0±7.56, X light =-22.52±12.61, p=0.520). For female single knockout mice, there was no significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-0.42 C±.14, X light=-0.35 C±.19, p=0.608) (Figure 4) or the mean decrease in locomotion (X dark=-5.80±7.10, X light = -6.20±15.04, p=0.975) (Figure 7). For male single knockout mice, there was also no significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-1.12 C±.33, X light=-0.77 C±.29, p=0.499) (Figure 3) or the mean decrease in locomotion(x dark=-28.14±14.60, X light =-45.85±19.77, p=0.449) (Figure 7). Among double knockout mice, the mean decrease in temperature (Figure 2) did vary significantly between the light and dark phase (X dark =-2.53 C±.55, X light =-1.16 C±.27, p=0.027), but their mean decrease in locomotion (Figure 5) did not vary significantly (X dark= ±14.14,X light=-71.10±10.78, p=0.911). For female double knockout mice, there was no significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-1.41 C±.48, X light=-1.01 C±.20, p=0.299) (Figure 4) or the mean decrease in locomotion (X dark=-56.66±15.59, X light = ±13.49, p=0.679) (Figure 7). For male double knockout mice, there was a significant difference between the dark and light phase in the mean decrease in body temperature (X dark=-3.54 C±.86, X light=-1.31 C±.49, p=0.049) (Figure 3), but no difference 9

18 in the mean decrease in locomotion (X dark=-80.40±23.15, X light = ±16.91, p=0.930) (Figure 7). Interestingly, the overall difference in temperature was in the opposite direction as for the C3H mice (that is, there was a smaller change in body temperature during the light phase), and this difference was mainly due to the male mice. 10

19 6 Body Temperature: Light v. Dark phase 5 decrease in C D-C3H L-C3H D-WT L-WT D-sKO L-sKO D-dKO L-dKO Figure 2: Dark/Light variations in decrease in body temperature. Note that positive values indicate a decrease in body temperature. The C3H and double knockout groups show significant variation between the light and dark phases, but the wild-type and single knockout mice do not. Overall, single knockouts and wild-types are less sensitive to nicotine s effects on body temperature. All error bars represent the standard error of the mean. 6 Male Body Temperature: Light v. Dark 5 decrease in C D-C3H L-C3H D-WT L-WT D-sKO L-sKO D-dKO L-dKO Figure 3: Dark/Light variations in male mice. Male C3H mice show significant variation between the light and dark phase. Double knockouts also show a significant variation, but in the opposite direction from the C3H mice. There is no significant difference between light and dark for the wild-type or single knockout mice. 11

20 6 Female Body Temperature: Light v. Dark 5 decrease in C D-C3H L-C3H D-WT L-WT D-sKO L-sKO D-dKO L-dKO Figure 4:Dark/Light variations in female mice. Female C3H mice show significant variation between the light and dark phase, but the wild-types, single knockouts and double knockouts do not. Locomotion: Light v. Dark phase decrease in locomotion D-C3H L-C3H D-WT L-WT D-sKO L-sKO D-dKO L-dKO Figure 5: Light/dark variations in locomotor depression. Note that positive values indicate a decrease in locomotion. Only C3H mice show a significant difference between light and dark phases. Overall, single knockouts and wildtypes are less sensitive to nicotine-induced locomotor depression. 12

21 Male Locomotion: Light v. Dark 140 decrease in locomotion D-C3H L-C3H D-WT L-WT D-sKO L-sKO D-dKO L-dKO Figure 6: Light/dark variations in locomotor depression in male mice. Male C3H mice show a significant variation in locomotion between the light and dark phase. There is a non-significant trend for smaller change in locomotion in the dark among the mutant mice. Female Locomotion: Light v. Dark decrease in locomotion D-C3H L-C3H D-WT L-WT D-sKO L-sKO D-dKO L-dKO Figure 7: Light/dark variations in locomotor depression in female mice. Female C3H mice show a significant variation between the light and dark phases, but there is no significant variation between the light and dark for the wild-types, single knockouts and double knockouts. 13

22 Locomotion & Body Temperature: 1.0 mg/kg nicotine 0400 hours relative clock time A two-way ANOVA was used to compare the effects of genotype and sex on body temperature and locomotion at 0400 h relative clock time and the least significant difference test (LSD) was used for post hoc analysis. There were significant main effects for genotype and sex on the change in body temperature and locomotion, as well as a significant interaction between genotype and sex for the change in body temperature. Genotype had a significant main effect on the decrease in body temperature (F=12.988, p=0.000) and locomotion (F=6.411, p=0.001) The LSD test showed that the double knockout mice (X =2.53 C±.55) had a significantly greater decrease in body temperature than the C3H ( =1.89 C, p=0.000), wild-type ( =2.15 C, p=0.000), and single knockout mice ( =1.86 C, p=0.000) (Figure 8). However, there were no significant differences between wild-type mice and the single knockout mice (p=0.450), the wild-types and the C3H mice (p=0.467) or the single knockouts and the C3H mice (p=0.944). For locomotion, double knockouts (X =69.15±14.14) also had significantly greater decrease in locomotion than the wild-type ( =64.15, p=0.000), single knockout ( =53.89, p=0.002), and C3H mice ( =56.36, p=0.001) (Figure 9). There were no significant difference between the wild-type mice and the single knockouts (p=0.534) or the wild-types and C3H mice (p=0.617). There was also no significant difference between the single knockout mice and the C3H mice (p=0.874). 14

23 6 Body temperature: 1.0 mg/kg nicotine 5 decrease in C C3H WT sko dko C3H WT sko dko Dark Light Figure 8: Decrease in body temperature after 1.0 mg/kg nicotine. Double knockouts have a much greater decrease in body temperature during the dark phase when compared to all other genotypes. During the light phase, double knockouts have a decrease in body temperature similar to the other mutant genotypes. 140 Locomotion: 1.0 mg/kg nicotine 120 decrease in locomotion Plot C3H WT sko dko C3H WT sko dko Dark Light Figure 9: Decrease in locomotion after 1.0 mg/kg nicotine. Double knockouts have similar decrease in locomotion during the light and dark, while the other genotypes have a greater decrease during the light phase. 15

24 There was also a significant main effect of sex on decrease in body temperature (F=7.949, p=0.000) and locomotion (F=6.411, p=0.001) at the 0400 h time point. The male mice had a significantly greater decrease in body temperature ( =0.73 C) and locomotion ( =24.08). Since there were only two levels of the variable (male and female), there was no post hoc analysis. There was also a significant interaction between sex and genotype on decrease in body temperature (p=0.031). This significant interaction reflects the fact that the difference between males and females is not the same size for each genotype. To further examine the effect of sex on decrease in body temperature and locomotion, we ran separate one-way ANOVAs on males and females. For change in body temperature among female mice, there was a significant effect of genotype on decrease in body temperature (F=3.626, p=0.022). Post-hoc analysis with the LSD test showed that the double knockout mice (X =1.41 C±.39) had a significantly greater decrease in body temperature than the C3H ( =0.88 C, p=0.016), wild-type ( =1.08 C, p=0.006) and single knockout mice ( = 0.99 C, p=0.010) (Figure 11). However, there were no significant differences between wild-type mice and the single knockout mice (p=0.791), the wild-types and the C3H mice (p=0.567) or the single knockouts and the C3H mice (p=0.760). For male mice, there was a significant effect of genotype on decrease in body temperature (F=9.859, p=0.000) and locomotion (F=3.398 p=0.028). The double knockout males (X =3.54 C±.37) had a significantly greater decrease in body temperature than the C3H ( = 2.79 C, p=0.000), wild-type ( = 3.12 C, p=0.000) and single knockout mice ( = 2.60 C, p=0.000) (Figure 10). There were no significant differences between wild-type mice and the 16

25 single knockout mice (p=0.447), the wild-types and the C3H mice (p=0.599) or the single knockouts and the C3H mice (p=0.776). There was also a significant effect genotype on locomotion among female mice (F=3.041, p=0.041). Double knockout females (X =56.66±16.86) also had significantly greater decrease in locomotion than the wild-type ( =66.33, p=0.009), single knockout ( ==50.86, p=0.037), and C3H mice ( =54.83, p=0.020) (Figure 13). There were no significant difference between the wild-type mice and the single knockouts (p=0.514) or the wild-types and C3H mice (p=0.613). There was also no significant difference between the single knockout mice and the C3H mice (p=0.857). For male mice, there was a significant effect of genotype on decrease in locomotion (F=3.398 p=0.028). Double knockout males also had significantly greater decrease in locomotion (X =80.40±15.99) than the wild-type ( =62.2, p=0.009), single knockout ( =54.62, p=0.023), and C3H mice ( =56.65, p=0.012) (Figure 12). There were no significant difference between the wild-type mice and the single knockouts (p=0.744) or the wild-types and C3H mice (p=0.797). There was also no significant difference between the single knockout mice and the C3H mice (p=0.927). 17

26 5 4 Male Body Temperature : 1.0 mg/kg nicotine decrease in C C3H WT sko dko Dark Phase Figure 10: Decrease in body temperature among male mice. The double knockouts had a significantly greater decrease in body temperature than all other genotypes during the dark phase. Since there was no significant effect of sex in the light phase, light phase data is not shown. 5 Female Body Temperature : 1.0 mg/kg nicotine 4 decrease in C C3H WT sko dko Dark Phase Figure 11: Decrease in body temperature among female mice. In the dark phase, double knockouts have significantly greater decrease than all other genotypes. (Light phase not shown) 18

27 Male Locomotion : 1.0 mg/kg nicotine 140 decrease in locomotion C3H WT sko dko Dark Phase Figure 12: Males decrease in locomotion. Double knockouts have a significantly greater decrease in the dark phase. (Light phase not shown) 140 Female Locomotion : 1.0 mg/kg nicotine 120 decrease in locomotion C3H WT sko dko Dark Phase Figure 13: Females decrease in locomotion. Double knockouts have a significantly greater decrease in locomotion in the dark phase. (Light phase not shown) 19

28 1600 hours relative clock time At the 1600 hours time point, two-way ANOVA revealed a significant main effect of genotype on decrease in body temperature (F=8.60, p=0.000) and locomotion (F=6.776, p=0.000); but there was no significant effect of sex on either body temperature (F=2.247, p=0.138) or locomotion (F=2.775, p=0.100). There was also no significant interaction between genotype and sex on decrease in body temperature (F=.167, p=0.918) or locomotion (F=1.347, p=0.265) The LSD test was used for post-hoc analysis. For body temperature, C3H mice had a significantly greater decrease in body temperature (X =2.24 C±.29) compared to the wild-type mice ( =1.49 C, p=0.000), single knockout mice ( =1.71 C, p=0.000), and double knockout mice ( =1.01 C, p=0.006) (Figure 8). There were no significant difference between wild-type mice and single knockouts (p=0.609) or between wild-type mice and double knockouts (p=0.258). There were also no significant differences between the single knockouts and the double knockouts (p=0.092). For locomotion at the 1600 time point, C3H mice (X =91.96±15.21) had a significantly greater decrease in locomotion than either wild-types ( =71.21, p=0.000) or single knockouts ( =69.43, p=0.001), but not double knockouts (p=0.415) (Figure 9). Double knockouts (X =77.33±10.73) also had a significantly greater decrease than single knockouts ( =54.80, p=0.009) and wild-types ( =56.85, p=0.008). Wild-types and single knockouts were not significantly different (p=0.935). 20

29 Locomotion & Body Temperature : 0.5 mg/kg nicotine At this dose we had 15 wild type mice (female=8), 12 single knockout mice (female=8) and 6 double knockout mice (female=1). We did not have sufficient numbers of C3H mice to test at this dose. Two-way ANOVA revealed no significant main effect for genotype at the 0400 h time point for either body temperature (F=.402, p=0.673) or for locomotion (F=.128, p=0.880) (Figure 14 and 15). There was also no significant main effect for sex for decrease in body temperature (F=1.473, p=0.235) and for locomotion (F=.277, p=0.603), and there was no significant interaction between genotype and sex for decrease in body temperature (F=.798, p=0.46) or for locomotion (F=.696, p=0.507). At the 1600 time point, there was no significant main effect for genotype and decrease in body temperature (F=1.053, p=0.365) or locomotion (F=.709, p=0.502) (Figure 14 and 15). There was a significant main effect for sex on the decrease in body temperature (F=4.182, p=0.052) and locomotion (F=5.751, p=0.025). However, when we ran separate one-way ANOVAs for males and females, there was no significant effect of genotype. For females at the 1600 time point, there was no significant difference between genotypes in decrease in body temperature (F=.240, p=0.79) or locomotion (F=2.167, p=0.151). For males at the 1600 time point, there was also no significant difference between genotypes in decrease in body temperature (F=.772, p=0.49) or locomotion (F=2.229, p=0.164). We then ran a one-way ANOVA on the combined data for males and females. There was no significant effect on body temperature (F=.280, p=0.758) or locomotion (F=.016, p=0.984), at the 0400 time point. At the 1600 time point there was no significant effect on body 21

30 temperature(f=2.121, p=0.140), but there was a significant effect of genotype on locomotion(f=4.902, p=0.016),. The LSD post hoc analysis showed that double knockouts (X = 26.00±11.00) had a greater decrease in body temperature than the single knockouts ( =53.9, p=0.004) or the wild-types ( =37.3, p=0.032). 22

31 3.5 Body temperature: 0.5 mg/kg nicotine 3.0 decrease in C WT sko dko WT sko dko Dark Light Figure 14: Decrease in body temperature after 0.5 mg/kg nicotine in the light and the dark. There are no significant differences among the groups in either the light or dark phase. 80 Locomotion: 0.5 mg/kg nicotine 60 decrease in locomotion Plot 1-40 WT sko dko WT sko dko Dark Light Figure 15: Change in locomotion after 0.5 mg/kg nicotine in the light and the dark. There is no difference between the genotypes in the dark, and in the light only the double knockouts show a significant decrease in locomotion. Note that negative values indicate an increase in locomotion. 23

32 Locomotion & Body Temperature: 1.5 mg/kg nicotine At this dose we had 16 wild type mice (female=8), 14 single knockout mice (female=9) and 12 double knockout mice (female=4). We did not have sufficient numbers of C3H mice to test at this dose. Two-way ANOVA at the 0400 time point showed no significant main effect for sex on the decrease in body temperature (F=2.365, p=0.133) or locomotion (F=.599, p=0.444). There was also no significant interaction for genotype and sex on body temperature (F=1.369, p=0.267) or locomotion (F=.577, p=0.567). At the 1600 time point, there was again no significant main effect of sex on the decrease in body temperature (F=.886, p=0.353) or locomotion (F=.550, p=0.463). There was significant interaction for genotype and sex on body temperature (F=3.882, p=0.030), but not for locomotion (F=.377, p=0.389). The significant interaction for the change in body temperature reflects a small sex difference among the double knockouts, where the males showed greater sensitivity than the females. Since there was no significant effect of sex on the decrease in body temperature or locomotion due to nicotine at either time point, we combined the sexes and ran a one-way ANOVA on the effects of genotype on change in body temperature and locomotor depression. One-way ANOVA at the 0400 h time point revealed a significant effect of both locomotion (F=3.202, p=0.051) and body temperature (F=4.415, p=0.019). The LSD test was used for post-hoc analysis. The double knockouts (X =87.75±17.91) had a significantly greater decrease in locomotion than the wild-types ( =45.63, p=0.017) (Figure 17). There was no significant difference between the single knockouts and double knockout mice (p=0.086), and no difference between the single knockouts and the wild-type mice (p=0.499). 24

33 For temperature, the double knockouts (X =3.00 C ±.50) had a significantly greater decrease in body temperature than the wild-types ( =1.52 C, p=0.015) and the single knockouts ( =1.68 C, p=0.010) (Figure 16). There was no significant difference between the single knockouts and the wild-type mice (p=0.779). One-way ANOVA at the 1600 time point revealed significant effects of locomotion (F=8.407, p=0.001) and temperature (F=7.231, p=0.002). The LSD test was used for post-hoc analysis. The double knockouts (X = ±9.68) had a significantly greater decrease in locomotion than the wild-types ( =64.51, p=0.000) or the single knockouts ( =49.29, p=0.005) (Figure 17). There was no significant difference between the single knockouts and the wild-types (p=0.344). The double knockouts (X =3.44 C ±.38) also had a significantly greater decrease in body temperature than the wild-types ( =2.15 C, p=0.000) or the single knockouts ( =1.14 C p=0.053), but there was no significant difference between the single knockouts and the wildtypes (p=0.077) (Figure 16). 25

34 5 Body temperature: 1.5 mg/kg nicotine 4 decrease in C WT sko dko WT sko dko Dark Light Figure 16: Decrease in body temperature in the light and the dark after 1.5 mg/kg nicotine. In both the light and the dark, the double knockouts had a significantly greater decrease in body temperature than the single knockouts or the wild-types. There was no difference between the single knockouts and the wild-types. 140 Locomotion: 1.5 mg/kg nicotine decrease in locomotion Plot WT sko dko WT sko dko Dark Light Figure 17: Decrease in locomotion in the light and the dark after 1.5 mg/kg nicotine. The double knockouts had a significantly greater decrease than the wild-types in both the light and the dark. The difference between single and double knockouts was only significant in the light. 26

35 Two-Bottle Preference For this protocol we had 16 wild type mice (female=10), 14 single knockout mice (female=11) and 18 double knockout mice (female=9). We did not have sufficient numbers of C3H mice to test at this dose. Total consumption of nicotine solution at each dose was used in this analysis. That is, the values for fluid intake are the totals for the four day period that the mice had access to each dose, not the averaged daily values. Repeated measures ANOVA of nicotine consumed by percent of total volume revealed a significant within-subjects linear effect of the solution s concentration (F=58.0, p=0.000), but not a significant quadratic effect (F=1.195, p=0.280). There was no significant interaction between concentration and genotype (F=2.189, p=0.124). There was a significant between-subjects effect (F=5.5,p=0.007). Nicotine consumption declined linearly as the concentration of the nicotine solution increased for all genotypes (Figure 18). Post-hoc analysis was performed using least significant difference test (LSD). There was a significant difference of nicotine consumption by percent of total consumption. Nicotine consumption by the double knockouts was consistently higher than either the single knockouts (p=0.002) or the wild-types (p=0.059). There was no statistically significant difference between the single knockouts and the wild-types (p=0.176). Repeated measures ANOVA was also performed on nicotine consumption measured as the dose of nicotine in milligrams per kilogram. This showed a significant within-subject quadratic effect of concentration (F=8.422, p=0.006) but no linear effect (F=0.163, p=0.662). There was no significant interaction between concentration and genotype (F=1.599, p=0.213). There was a significant between-subjects effect (F=4.536, p=0.016). Nicotine consumption as 27

36 measured by mg/kg showed a quadratic trend, peaking at the 50 µg/ml concentration (Figure 19). Post-hoc analysis was performed using LSD test. Nicotine consumption by the double knockouts was consistently higher than either the single knockouts (p=.006) or the wild-types (p=.038). There was no statistically significant difference between the single knockouts and the wild-types (p=.444). 28

37 mg /kg Nicotine Consumption: % of total consumption volume % of total consumption µg/ml 50 µg/ml 100 µg/ml Figure 18: Nicotine consumption measured as percentage of total consumption. Double knockouts consistently drink a greater percentage of their fluid intake from the nicotine solution than either wild-types or single knockouts at the 50 and 100 μg/ml concentrations. At 25 μg/ml, double knockouts and wild-type are similar, and both have greater nicotine consumption than the single knockouts. 5 4 Nicotine Consumption (mg/kg) mg/kg µg/ml 50 µg/ml 100 µg/ml Figure 19: Nicotine consumption measured as nicotine dose in milligrams/kilogram. Double knockouts take drink a higher dose of nicotine at the 50 and 100 μg/ml concentrations. 29

38 25 ug/l %nic 50 ug/l %nic 100 ug/l %nic 25 ug/l nic mg/kg 50 ug/l nic mg/kg 100 ug/l nic mg/kg Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total ANOVA of Nicotine Consumption Sum of Squares df Mean Square F Sig Table 1: One-way ANOVA comparisons of all three genotypes at each nicotine solution concentration and each metric for nicotine consumption. All comparisons are significant. 30

39 Two-way ANOVA revealed no significant effect of sex for any dose using either metric, so the sexes were combined and one-way ANOVA was also used to measure nicotine consumption at each concentration of the nicotine solution, and the test of LSD used for post-hoc analysis. One-way ANOVA revealed a significant effect of genotype at all nicotine concentrations, whether or not consumption was measured as percent of total volume or as dose in mg/kg (Table 1). Post-hoc analysis showed that at the 25 µg/ml nicotine concentration, percent nicotine consumption for the double knockouts (X =38.8% ±4.6%) was significantly greater than for the single knockouts ( =21.7%, p=0.003), but not different than the consumption for the wild-types ( =5.9%, p=0.376). There was also a significant difference in percent consumption between wild-types and single knockouts ( =15.7%, p=0.032). When measured as dose in mg/kg, double knockouts (X =2.10 ±.31) again had significantly greater consumption than single knockouts ( =1.09, p=0.008), but no difference between double knockouts and wild-types (p=0.270) or between wild-types and single knockouts (p=0.103) At the 50 µg/ml concentration, percent consumption for double knockouts (X =30.9% ±5.5%) was significantly greater than the single knockouts ( =18.0%, p=0.007), and greater than the wild-types ( =13.3%, p=0.035). But there was no significant difference between the wildtypes and the single knockouts (p=0.484). For nicotine consumption as dose in mg/kg, double knockouts (X =3.211 ±.53) still drank significantly more than single knockouts ( =1.70, p=0.014) or the wild-types ( =1.37, p=0.037), but there was no significant difference between the wild-types and the single knockouts (p=.637). 31

40 At the 100 µg/ml concentration, percent consumption for the double knockouts (X =14.9% ±5.1) was significantly greater than the single knockouts ( =10.9%, p=0.028) or the wild-types ( =9.5%, p=0.047). But there was no significant difference between the wild-types and the single knockouts (p=0.777). For nicotine consumption as dose in mg/kg, double knockouts (X =3.10 ±1.00) still drank significantly more than single knockouts ( =2.22, p=0.026) or the wild-types ( =1.83, p=0.056), but there was no significant difference between the wild-types and the single knockouts (p=0.691). Nicotine Preference by Sex To check for the potential influence of sex on nicotine consumption, we performed a twoway ANOVA of sex and genotype. There was no significant effect of sex on nicotine consumption (F= , p= ), so no post-hoc analysis was performed. 32

41 Discussion Locomotion & Body Temperature: Light v. Dark phase (1.0 mg/kg nicotine) C3H mice vary significantly in their reaction to nicotine during the light and dark phases of the 24-hour circadian cycle. Specifically, the mean decrease in body temperature and mean decrease in locomotion was significantly greater in the light phase (1600 h), when melatonin and melatonin receptor levels are at their lowest. Among our genetically modified mice, neither the wild-type nor the single knockout mice had a significant difference in their reaction to nicotine when comparing the light phase and the dark phase. There was a general trend towards greater decrease in body temperature and locomotor depression in the light phase, but this was not significant. However, the wild-type and single-knockout mutants also had a greatly attenuated response to nicotine overall, so it is possible that any significant difference between light and dark was not detectable. Double knockout mice did not have a significant difference in locomotor depression, but they did have a significant difference in their decrease in body temperature when the light phase was compared with the dark phase. Interestingly, this difference in body temperature was in the opposite direction of the C3H mice; that is, double knockout mice had a greater decrease in body temperature during the dark phase than during the light phase. This may indicate that melatonin receptors are more involved in loss of thermoregulation after nicotine challenge or that the animals have a greater sensitivity to nicotine at night which is normally masked by the melatonin receptors. The wild-type melatonin knockout mice did not have a similar light/dark variation similar to the C3H mice, despite the knockout genotype being developed on a C3H background. Specifically, the wild-type mice have a greatly attenuated response to nicotine in both the light and dark. This may have occurred for a couple of reasons. First, the C3H mice colony used in 33

42 this study had been maintained at IBG in Boulder, Colorado for 40 years, which is sufficient time for the development of a sub-strain of C3H mice (C3H/IBG) different from the C3H colony used to develop the knockout genotype. Second, wild-type mice are the product of a lineage which involves the C3H/HeN substrain. Although there should be no significant differences among the substrains, it is possible that there are some residual effects of the genetic modification process that interfere with the wild-types reaction to nicotine or expression of melatonin. However, subsequent analysis showed that at each time point, the wild-type and C3H mice were statistically similar. Also, the sex difference in locomotion and body temperature appears to be restricted to mutant genotype mice. This may account for some of the difference between the C3H mice and the wild-type melatonin knockouts. 1.0 mg/kg nicotine : 0400 h relative clock time During the dark phase, double knockouts had significantly greater decreases in both locomotion and body temperature than any other genotype, consistent with our hypothesis that increased melatonin receptor levels at night attenuate the response to nicotine. Among the C3H mice, wild-type mice and single knockout mice, there were no significant differences in locomotor depression or decrease in body temperature at This suggests two things; first, that the wild-type mice are an acceptable control genotype, since they do not vary significantly from the C3H mice. Second, since the single knockout mice behave similarly to the wild-type mice, either the melatonin 1a receptor is not involved in circadian regulation of nicotine response, or the single knockout animals are able to compensate for the lack of the melatonin 1a receptor, probably by upregulating melatonin 1b receptor expression. We also considered the effects of sex on the mice s response to nicotine. In all four genotypes, male mice had a greater decrease in body temperature and locomotor activity than the 34