Published: Vol 8, Iss 20, Oct 20, 2018 DOI: 10.21769/BioProtoc.3061 Views: 5985
Reviewed by: Edgar Soria-GomezMohammed Mostafizur RahmanOscar Prospero
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Abstract
The alcohol preference model is one of the most widely used animal models relevant to alcoholism. Stressors increase alcohol consumption. Here we present a protocol for a rapid and useful tool to test alcohol preference and stress-induced alcohol consumption in mice. In this model, animals are given two bottles, one with a diluted solution of ethanol in water, and the other with tap water. Consumption from each bottle is monitored over a 24-h period over several days to assess the animal’s relative preference for the ethanol solution over water. In the second phase, animals are stressed by restraining them for an hour daily and their subsequent preference of tap water or the ethanol solution is evaluated. Preference is measured by the volume and/or weight or liquid consumed daily, which is then converted to a preference ratio. The alcohol preference model was combined with the conditioned place preference paradigm to determine alcohol conditioning and preference following the deletion of CB2 cannabinoid receptors in dopaminergic neurons in the DAT-Cnr2 Cre-recombinant conditional knockout (cKO) mice in comparison with the wild-type control mice.
Keywords: AlcoholBackground
Many aspects of alcoholism and alcohol consumption can be studied through animal models. Alcohol induces positive reinforcement, and animals can seek alcohol and even work for it. However, alcohol can also be a negative reinforcement, since it is capable of reducing anxiety. No animal model is able to duplicate the complex features of alcoholism. Oral ethanol self-administration is widely used for examining specific aspects of behavior and physiology relevant for understanding alcoholism (Mardones and Segovia-Riquelme, 1983; Cunningham et al., 2000). Mice can be genetically manipulated at cell type specific levels and therefore are valuable for research into the cell type specific genetic determinants of alcoholism.
The alcohol preference model is one of the most widely used animal models relevant to alcoholism. This model meets important criterion, which is that the ethanol should be self-administered orally (Cicero, 1980; Crabbe et al., 2010). An animal’s genotype exerts a strong influence on self-administration in this model. Some mouse strains, like the inbred strain of mouse C57BL/6J (Rhodes et al., 2005), present a genetically influenced high preference for ethanol and they voluntarily consume it orally (Yoneyama et al., 2008; Barkley-Levenson and Crabbe, 2012). The conditioned place paradigm (CPP) is widely used to explore the effects of addictive substances including alcohol, taking advantage of learned associations. Therefore, alcohol CPP measures the association of alcohol with a particular environment to determine whether mice can acquire alcohol CPP.
Stress can interact with ongoing ethanol consumption to trigger increased intake (e.g., self-medicating behavior), thereby increasing initial susceptibility to alcohol use disorders. Among the stressors, a restraint model of acute and chronic stress can increase ethanol consumption (Yang et al., 2008).
New advances and accumulating evidence support a role for the endocannabinoid system in the effects of alcohol. The endocannabinoid system consists of two cannabinoid receptors, CB1Rs and CB2Rs, with endocannabinoids and the enzymes for the biosynthesis and inactivation of the endocannabinoids. Our goal here was to summarize the protocol used to measure alcohol preference in combination with stress-induced alcohol consumption. We also provide evidence that the endocannabinoid system plays a role in alcohol preference following dopaminergic neuron specific deletion of CB2Rs in the mouse model (Liu et al., 2017).
Materials and Reagents
Equipment
Software
Procedure
All experiments were conducted during dark phase, under dim light illumination.
Data analysis
All data are transcribed in a spreadsheet. Set up a column for the raw weight data and next to it a column for the corrected weight data. The corrected weight data is the amount of spillage (of water and alcohol) each day from the control cage. This number will be subtracted from the raw weight data. Each day, the spillage amount must be subtracted from the raw water and alcohol weight data to determine the corrected weight.
To calculate the amount of alcohol or water consumed per day, the bottle weights were averaged for each day. These values were then subtracted from the averages of the bottle weights for each group on that day. Subtracting the new bottle weight from that of the previous day gave the amount of alcohol or water drank by the mice overnight.
Day 0 is when the bottles were initially filled with 150 ml of water/alcohol and therefore the consumption for that day was 0.
The alcohol preference ratio was determined by dividing the amount of alcohol consumed by total liquid (alcohol + water) consumed.
The activity monitor recorded time spent in each of the compartments, using the photo electric beam break counts. The time that each mouse spent in the compartment was recorded and the CPP score was defined as the time spent in the alcohol paired compartment minus the time spent in the saline-paired compartment during the CPP test. The activity monitor software (Med Associates, St. Albans, VT) was used for automated data collection.
Representative data: Figure 4 is representative data for Alcohol Preference Test. In this test, the mice were individually housed and habituated with two fluid bottles available for three days. We used two groups: 12 male C57/6J mice as wild-type and 12 male DAT-Cnr2 conditional knockout (cKO) mice that do not express the CB2R in midbrain dopamine neurons. On the day of the experiment, all the animals were weighed. One bottle was filled with 150 ml of tap water and the other was filled with 150 ml of 16% alcohol solution. They were weighed with the tops on and placed on top of the cages (labeled correctly). For the alcohol preference test, the bottles were weighed for each animal for five consecutive days at 10 AM. The positions of the bottles in the different cages were randomized with respect to which side of the cages they were placed. In the second part of the experiment, each group was then split into two groups. One group (n = 6) was stressed by putting them in a 50 ml tube for one hour each day for 5 consecutive days. The rest of the animals remained in their cages. All the bottles were weighed for each animal for five consecutive days at the same time, and all the bottles were placed on top of the cages at 11 AM. In all the experiments, the ratio of alcohol to water consumed and the total fluid consumption were calculated to obtain a preference ratio. The alcohol preference ratio was determined by dividing the amount of alcohol consumed by total fluid (alcohol + water) consumed, with and without the sub-acute stress. The wild-type mice preferred alcohol over water, as evident by the significant increase (as determined by Repeated Measurement ANOVA). The DAT-Cnr2 cKO mice (mice did not show an alcohol-CPP induction, Figure 5), and do not show a significant increase of the preference for alcohol (Figure 4). Wild-type mice preferred more alcohol than the DAT-Cnr2 mice during the 5-day test with acute stress (as determined by Student’s t-test comparison between genotypes). These results suggest that the DAT-Cnr2 KO mice are resistant to alcohol consumption even in acute stress conditions. This supports the previous report that CB2 receptor agonists is involved in the development and enhancement of alcohol preference in stressed but not in non-stressed control mice (Ishiguro et al., 2007; Onaivi et al., 2008).
Figure 4. Preference ratio for alcohol over water in normal conditions (A) and in response to an acute stress protocol (B) exhibited by wild-type mice (white circles) and DAT-Cnr2 cKO mice (black circles) during 5 consecutive days. Data presented as the Mean ± standard error of the mean (SEM) daily consumption ratio over 5 days. Significant difference between genotypes paired t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 5. Deletion of CB2Rs in dopamine neurons (DAT-Cnr2 cKO) mice and alcohol preference. 8% alcohol-induced CPP (P < 0.05) in wild-type mice but the DAT-Cnr2 cKO mice did not show significant difference in post-conditioning phase between alcohol and saline.
Notes
Animal considerations:
Acknowledgments
This protocol is in memory of our co-author, Norman Schanz, whose sudden and tragic loss will be difficult to replace in the animal laboratory. This research was supported by William Paterson University. ACA was supported by the Mexican National Council of Science and technology (CONACYT # CVU332502/232728). HI was supported by Public Interest Trust Research Aid Fund for Stress-Related Diseases (with Commemoration of Imai Kimi), KAKENHI (18023009, 20023006, and 20390098). ESO was supported by NIH grant DA032890. QRL is supported by IRP/NIA/NIH. We thank the Dean, Dr. Venkat Sharma, of the College of Science and Health at William Paterson University for student worker support in maintaining our mice in the animal laboratory. We are also thankful for the technical assistant of Sneha Tammareddy, Eugene Dennis, Branden Sanabria, Paola Velandia, Steve Gross, and Monika Chung while working in the laboratory of ESO. This protocol and figures are adapted from our previous work (Liu et al., 2017).
Competing interests
The authors have no conflicts of interest to declare.
Ethics
The experimental procedures followed the Guide for the Care and Use of Laboratory Animals and were approved by William Paterson University Animal Care and Use Committee (IACUC).
References
Article Information
Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Canseco-Alba, A., Schanz, N., Ishiguro, H., Liu, Q. and Onaivi, E. S. (2018). Behavioral Evaluation of Seeking and Preference of Alcohol in Mice Subjected to Stress. Bio-protocol 8(20): e3061. DOI: 10.21769/BioProtoc.3061.
Category
Neuroscience > Behavioral neuroscience > Animal model
Neuroscience > Nervous system disorders > Animal model
Molecular Biology > RNA > RNA detection
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