Supplementary Data to Ozburn et al. NPP Submission
Methods and Materials
Drugs. Ethanol (Aaper, Shelbyville, KT, USA), ketamine hydrochloride (Fort
Dodge Animal Health, Fort Dodge, IA, USA), xylazine hydrochloride (Akom Inc.,
Decator, IL, USA) or pentobarbital sodium (Ovation Pharmaceuticals Inc.,
Deerfield, IL, USA) were dissolved in 0.9% saline and injected intraperitoneally
(i.p.) in a volume of 0.2 ml per 10 g of body weight.
Continuous Access Two-bottle Choice.
Ethanol - As described in Ozburn et al. (2010), male mice were habituated to
individual housing and sipper bottles for one week prior to the start of the
experiment. Mice were offered water and 3% ethanol (v/v in tap water) for 2
days. After 3% ethanol, escalating concentrations (up to 21%) were offered
versus water, 2 days each. Fluid intake was measured daily. Mice were weighed
every four days. Ethanol preference (mL ethanol solution consumed/mL total fluid
consumed), ethanol consumption (g pure ethanol/kg body weight/day), and total
fluid consumption were measured. n=9/genotype.
Quinine - As described in Blednov et al. (2010), male and female mice were
habituated to individual housing and sipper bottles for one week prior to the start
of the experiment. Mice were serially offered offered 0.03 and 0.06 mM quinine
hemisulfate (in tap water) for 4 days each. Fluid intake was measured daily. Mice
were weighed every four days. Quinine preference (mL quinine solution
consumed/mL total fluid consumed) was measured. n=9-11/sex/genotype.
Conditioned Taste Aversion. As described in Ozburn et al. (2010), mice were
adapted to a water-restriction schedule (2 hr water/day; ZT2-4) over 7 days. At
48 hr intervals over the next 12 days, mice received 1 hr access to a solution of
saccharin (0.15% wt/vol) at ZT2. After 1 hr access to saccharin, mice received
injections of saline or ethanol (2 g/kg). All mice received 1 hr access to tap water
4 hr after injections to prevent dehydration. 2 hr access to tap water was given
during intervening days. n=12/group/genotype.
Acute Functional Tolerance. Testing was carried out similar to the protocol
described by Erwin and Dietrich (1996). At ZT6, mice were placed on a rotarod
(fixed 5 RPM) and required to stay on rotarod for 1 minute before testing. Mice
were then injected with ethanol (1.75 g/kg) and placed on rotarod every 5
minutes until they could stay on rotarod for 1 minute. At this time, retro-orbital
blood samples were collected (BEC1), and mice were injected with ethanol (1.75
g/kg) a second time and then placed on the rotarod every 5 minutes until they
could stay on rotarod for 1 minute. Upon the second rotarod recovery, another
blood sample was taken (BEC2). n=6/genotype.
Ethanol Clearance. As described in Ozburn et al. (2010), mice were injected
with ethanol (4 g/kg) at ZT 4 or ZT16 and rates of ethanol clearance were
determined using a spectrophotometric enzyme assay. Blood samples (50 uL)
were taken from retro-orbital sinus (at 30, 60, 120, 180, and 240 min post
injection), added to 2 mL 3% perchloric acid, and centrifuged for 10 min at 1000 x
g. Resulting supernatants were used to determine blood ethanol concentration
(BEC) using an alcohol dehydrogenase enzyme assay. n=3-4/ZT/genotype.
Statistical Analysis. All data are expressed as mean ± SEM. Significance for
the acute functional tolerance assay was determined by Student’s t-test. Limited
access drinking data, CPP, CTA, and ethanol metabolism data were analyzed by
two-way analysis of variance (ANOVA). Repeated measures were applied where
appropriate. In all experiments, p < 0.05 is considered significant.
Results
Continuous Access Two-bottle Choice.
Ethanol – Male ClockΔ19 exhibit a strong trend toward increased ethanol
preference.
To determine if functional CLOCK is important for voluntary ethanol drinking in
male mice, we measured ethanol preference and consumption in ClockΔ19 and
WT littermates using the continuous access two-bottle choice paradigm.
ClockΔ19 male mice exhibited no difference in ethanol intake and a strong trend
for increased ethanol preference that was accompanied by reduced total fluid
consumption (Fig. 1a,b,c; Ethanol consumption: genotype x concentration
interaction – F(6,96)=0.63, p=0.71, main effect of genotype – F(1,16)=0.96,
p=0.34, main effect of concentration – F(6,96)=5.4, p<0.0001; Ethanol
preference: genotype x concentration – F(6,96)=0.61, p=0.27, main effect of
genotype – F(1,16)=3.86, p=0.06, main effect of concentration – F(6,96)=7.35,
p<0.0001; Total Intake: genotype x concentration – F(6,96)=1.21, p=0.30, main
effect of genotype – F(1,16)=15.19, p<0.001, main effect of concentration –
F(6,96)=5.59, p<0.0001).
Quinine – ClockΔ19 exhibit similar quinine preference.
Preference for non-alcohol tastants are important determine for mutant and WT
comparisons and can relate to ethanol preference. For instance, some studies
have revealed a positive correlation between preference for sweet solutions and
alcohol in mice (Blednov et al., 2012), while others have reported no relationship
(Blednov et al., 2007). It has been previously shown that ClockΔ19 mice exhibit a
higher preference for the sweet tastant, sucrose (6% average increase in
preference; Roybal et al. 2005). To determine if functional CLOCK is also
important for the bitter tastant preference, we measured quinine preference in
ClockΔ19 and WT littermates using the continuous access two-bottle choice
paradigm. ClockΔ19 and WT mice exhibited similar quinine preference and both
genotypes exhibited decreased preference at the higher quinine concentration
(Fig. 2a,b; Females - genotype x concentration interaction – n/s, main effect of
genotype – n/s, main effect of concentration – F(1,18)=19.56, p<0.001; Males genotype x concentration interaction – n/s, main effect of genotype – n/s, main
effect of concentration – F(1,18)=19.99, p<0.001).
ClockΔ19 and WT littermates display similar ethanol aversion, acute
functional tolerance, and clearance rates.
We have previously shown that Clock has a role in the neurobiological processes
involved in both the behavioral response to cocaine and the incentive motivation
for cocaine, prompting a more rigorous examination of ethanol responses and
related phenotypes in ClockΔ19 and WT littermates. Differences in ethanol intake
can be due to differences in the aversive properties of ethanol. Ethanol-induced
conditioned taste aversion (CTA) is often used as a measure of the aversive
properties of drugs of abuse. CTA strongly and negatively correlates with ethanol
intake (Green and Grahame, 2007). To examine the possibility that differences in
ethanol intake could be due to differential responses to the aversive effects of
ethanol, we measured ethanol-induced CTA and hypothesized reduced CTA in
ClockΔ19 mice. ClockΔ19 and WT mice expressed CTA to a similar extent
suggesting a similar sensitivity to the aversive properties of ethanol (Fig. 4a;
main effect of treatment – F(1,44)=52.05, p<0.0001). Increased ethanol drinking
could be due to increased development of acute functional tolerance, as well as
increased ethanol clearance. However, ClockΔ19 and WT mice acquire similar
acute functional tolerance and exhibit similar rates of ethanol clearance at both
times of day tested (Fig. 4b,c). Since ClockΔ19 mice exhibited increased ethanol
intake not accompanied by obvious correlations with other ethanol-related
behaviors, we further examined their pharmacological responses to other drugs
of abuse with sedative properties.
Supplemental Figures and Legends
Supplemental Figure 1. Male Clock∆19 mice exhibit a strong trend toward
increased ethanol preference. (A) Ethanol consumption (B) Ethanol preference
(C) Total fluid intake.
Supplemental Figure 2. Clock∆19 and WT mice exhibit a similar taste aversion to
quinine. (A) Female quinine solution preference. (B) Male quinine solution
preference.
Supplemental Figure 3. Clock∆19 and WT mice exhibit similar behavioral and
metabolic responses to ethanol. (A) Ethanol-induced conditioned taste aversion
to saccharin (B) Ethanol-induced acute functional tolerance (C) Rates of ethanol
clearance.
References
Blednov YA, Mayfield RD, Belknap J, Harris RA. (2012) Behavioral actions of
alcohol: phenotypic relations from multivariate analysis of mutant mouse data.
Genes Brain Behav. 11:424-435.
Blednov YA, Cravatt BF, Boehm SL 2nd, Walker D, Harris RA (2007) Role of
endocannabinoids in alcohol consumption and intoxication: studies of mice
lacking fatty acid amide hydrolase. Neuropsychopharmacology 32:1570-1582.
Blednov YA, Ozburn AR, Walker D, Ahmed S, Belknap JK, Harris RA (2010)
Hybrid mice as genetic models of high alcohol consumption. Behav Genet 40:93110.
Erwin VG, Deitrich RA (1996) Genetic selection and characterization of mouse
lines for acute functional tolerance to ethanol. J Pharmacol Exp Ther 279:13101317.
Green AS, Grahame NJ (2007) Ethanol drinking in rodents: is free-choice
drinking related to the reinforcing effects of ethanol? Alcohol 42:1-11.
McClung CA, Sidiropoulou K, Vitaterna M, Takahashi JS, White FJ, Cooper DC,
Nestler EJ (2005) Regulation of dopaminergic transmission and cocaine reward
by the Clock gene. Proc Natl Acad Sci USA 102:9377-9381.
Ozburn AR, Harris RA, Blednov YA. (2010) Behavioral differences between
C57BL/6J x FVB/NJ and C57BL/6J x NZB/B1NJ F1 hybrid mice: relation to
control of ethanol intake. Behav Genet 40:551-563.
Ozburn AR, Larson EB, Self DW, McClung CA (2012) Cocaine selfadministration behaviors in ClockΔ19 mice. Psychopharmacology 223:169-177.