Zachary Gardner and Scott Steffensen, Department of Psychology and Center for Neuroscience

Introduction

Substance abuse is a prolific problem with serious consequences. Specifically, alcohol abuse is the third leading cause of preventable mortality in the world (Mokdad et al., 2004). Each year the far-reaching effects of alcohol abuse cost the United States several hundred billion dollars annually (Office of National Policy, 2004). Treatment for individuals who suffer from this crippling disease is limited. In order to develop effective treatments we need to know more about the mechanisms of addiction and the changes that occur in the brain as a person becomes addicted.

Dopamine (DA) release within the nucleus accumbens (NAc) of the brain is associated with feelings of pleasure and reward. DA releasing neurons are regulated by gamma-amminobutyric acid (GABA) neurons in the ventral tegmental area (VTA). GABAA receptors are suspected to have binding sites for EtOH. Previous research has shown that DA release in mice that lack a δ subunit in their GABA neurons, termed delta knockout mice (δ-/-), respond differently than wild type (WT) mice do when exposed to EtOH. This evidence suggests that the δ subunit may play a role in EtOH addiction. The purpose of this project was and continues to be to determine whether δ-/- mice continue to display a lack of response under chronic EtOH conditions.

Methodology

Drink in the Dark: In order to determine EtOH dependence the drink in the dark protocol was employed. At the age of five weeks, both WT mice and GAD GFP mice underwent initial (DID) training. The mice were placed in a completely dark room into a chamber containing two bottles, one of dilluted EtOH (20%) and one of water. The amount of each consumed was recorded after a two-hour period. After five days of initial training, the mice were placed in a vapor chamber for four consecutive days and exposed to EtOH vapor for 16 hours day; other mice in chambers were exposed to air as a sham control group. After this four-day period the mice were removed from the vapor chamber and entered into a period of withdrawal. After three days of withdrawal, the mice underwent another round of DID. This process was repeated and water and EtOH consumption were measured and normalized. After inconclusive results this paradigm was altered. Mice underwent DID experimentation after 2 and 3 weeks of EtOH exposure in chambers with no pervious DID training. A third protocol was also introduced a third set of mice wherein mice underwent DID protocol daily without any prior EtOH exposure for two weeks.

Results

A pivotal part of the proposed experiment is the ability to determine EtOH dependence in mice. After an initial round of EtOH vapor exposure and DID protocol we were unable to determine dependence based on ethanol preference alone (Figure 1). We decided to change the paradigm so that mice were exposed to two weeks of EtOH in the vapor chambers followed by the DID protocol with no previous training. This protocol also failed to provide significant results. A third protocol in which three weeks of exposure in chambers followed by DID protocol without prior training also failed to demonstrate dependence. We also became aware that the DID protocol may induce preference in mice so a fourth paradigm was introduced in which preference for EtOH was measured in naïve mice with no prior EtOH exposure was measured using the DID protocol daily for two weeks (Figure 2). Dependence was not demonstrated in experiment either. No significant increase in EtOH drinking was or change in preference over time occurred.

The inconclusive nature of these experiments was not without significance. The DID protocol has been found to be an effective means of demonstrating EtOH preference, while allowing an animal to demonstrate drug seeking behavior. We were able to demonstrate EtOH preference in our electrophysiologic and neurochemical experiments, confirming prior assumptions. It has become clear over several months of testing that the widely used DID protocol is not an effective means of determining dependence

Discussion

The DID protocol is a lengthy complicated assay. Limited space and experimental equipment prevented us from performing multiple experiments at a time. Each protocol calls for several weeks of EtOH exposure before DID can be run. We also learned that 100% EtOH originally diluted in the experiment contains a taste aversive chemical. We switched to 95% EtOH which is free of this substance. We also confirmed in our experimentation that the white strain of mice (CD-1) used is less sensitive to EtOH and could not be used, while a black strain (C57BL6) prefers EtOH. Variable air pressure within the EtOH chambers themselves may have also affected our results.

Conclusion

After several months of careful experimentation and after exhausting all feasible ways in which to determine dependence by employing the DID protocol, we have concluded that it is not an effective means for determining EtOH dependence in mice. The DID protocol is an effective experimental protocol for determining preference. It allows the animal to demonstrate drug-seeking behavior. However it has become clear that preference does not necessarily equate to dependence. Distinct withdrawal symptoms, which are often subjective, are currently one of the only means of determining dependence effectively. A more effective assay must be developed in order to determine dependence so that the role of the δ subunit, as it pertains to EtOH addiction, can be further elucidated.

References

• Mokdad AH, Marks JS, Stroup DF, Gerberding JL (2004) Actual causes of death in the United States, 2000. JAMA : the journal of the American Medical Association 291:1238-1245.
• Office of National Drug Control Policy (2004). The Economic Costs of Drug Abuse in the United States, 1992-2002. Washington, DC: Executive Office of the President. (Publication No. 207303)