Changes to genetic material in the brain may help induce the anxiety that is characteristic of alcohol withdrawal, according to a new study supported by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), part of the National Institutes of Health (NIH). The finding points to possible therapies to prevent withdrawal-related anxiety, a driving force behind alcohol use among dependent individuals.

“The novel mechanism described in this study is an intriguing contribution to efforts aimed at defining the complex molecular processes that underlie alcohol abuse and dependence,” said NIAAA Deputy Director Kenneth Warren, Ph.D.

Previous studies have implicated a brain structure known as the amygdala in anxiety and alcohol-drinking behaviors. Other studies have shown that chemical modifications to chromatin, the complex of DNA and proteins within every cell nucleus, can influence the expression of genes and thus may affect disease processes.  Such modifications to DNA or its associated proteins that do not affect the DNA sequence are collectively referred to as epigenetic changes. One typical chromatin modification involves the addition or removal of acetyl groups – common components of many molecules in biology – to or from chromatin proteins called histones.

In the current study, scientists found evidence that the removal of acetyl groups (deacetylation) from histones in amygdala chromatin was associated with anxiety brought on by alcohol withdrawal.  A report of the study, led by Subhash C. Pandey, Ph.D., professor and director of neuroscience alcoholism research in the Department of Psychiatry at the University of Illinois at Chicago and Jesse Brown VA Medical Center in Chicago, appears in the April 2, 2008 issue of the Journal of Neuroscience.

The researchers modeled long-term alcohol use by maintaining adult rats on a liquid diet containing alcohol.  They found that when alcohol was withdrawn from the rats’ diet, the animals exhibited significantly higher anxiety-like behaviors than did rats that had been fed a non-alcohol diet.  One means of measuring anxiety used by the researchers was an apparatus known as an elevated plus-maze, which consists of two open arms and two closed arms connected to a central platform. Anxiety is gauged as a function of the amount of time a rat spends in the closed versus the open arms of the maze during a  five minute testing period — the greater an animal’s level of anxiety, the less open-arm activity it displays.

The activity of histone deacetylases (HDAC), enzymes that remove acetyl groups from histone molecules, was increased in the amygdala of alcohol-fed animals during withdrawal, and was reflected in a reduction in acetylation of histone proteins H3 and H4 in the central and lateral areas of the amygdala. They also found that alcohol withdrawal led to reduced levels of neuropeptide Y (NPY), a molecule known to play a role in anxiety and alcohol-drinking behaviors, in the amygdala of the alcohol-fed rats.  By blocking HDAC activity with an HDAC inhibitor known as trichostatin A, the researchers prevented the development of alcohol withdrawal-related anxiety, and restored levels of histone acetylation and NPY.

“This is the first direct evidence implicating the HDAC-induced chromatin remodeling in the amygdala as a major factor in the molecular processes of alcohol addiction and further suggests that HDAC inhibitors may be potential therapeutic agents in treating alcoholism,” said Dr. Pandey.

"This study demonstrates that alcohol exposure can cause epigenetic changes to chromatin, altering gene expression and behavior," added Antonio Noronha, Ph.D., director of NIAAA’s Division of Neuroscience and Behavior.

The researchers also investigated the potential role played by chromatin modifications in the anxiety-reducing effect that occurs with exposure to low levels of alcohol.  Rats injected with a one-time dose of alcohol exhibited less anxiety than control animals.  And in contrast to the increased HDAC activity observed during withdrawal-related anxiety, HDAC activity was inhibited during one-time alcohol exposure, while histone acetylation and NPY expression in the amygdala was increased.

Dr. Pandey noted that another class of enzymes – histone acetyltransferases (HATs) -- also regulate the addition of acetyl groups to histone molecules. He and his colleagues found that levels of a protein known as CREB binding protein (CBP), which contains HAT enzymatic activity, were increased by single-dose alcohol exposure but were decreased during ethanol withdrawal in alcohol dependent animals.

“Thus, both HDAC and HAT may be involved in the dynamic process of chromatin remodeling in the amygdala during alcoholism,” he said.  “Future studies will examine chromatin remodeling-linked epigenetic mechanisms of changes in expression of genes in the amygdala or other brain regions during alcohol tolerance and dependence.”