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Volume 18, Number 4, December 2007
Criswell and Colleagues Illuminate
the Mystery of Alcohol Actions
Scientists at UNC’s Bowles Center for Alcohol Studies spend their days elucidating the physiological and behavioral effects of alcohol. By showing how alcohol works, they advance the development of interventions to prevent and treat alcohol abuse and alcoholism, which directly affects more than 15% of individuals in the United States. Bowles Center Professor Dr. Hugh Criswell has devoted years to studying one of the great puzzles known to alcohol researchers: exactly how does alcohol affect the brain’s GABAergic system? GABA (gamma-amino-butyric acid) is the brain’s major inhibitory neurotransmitter. Interventions that increase GABAergic neurotransmission depress central nervous system activity and cause corresponding behavioral effects such as sedation. The central nervous system depression associated with alcohol ingestion has long been presumed to be caused by alcohol enhancement of GABAergic neurotransmission. While alcohol’s ability to influence the GABAergic system has been established for decades, the mechanisms by which alcohol increases GABAergic function have remained elusive.
Criswell Lab (Left to Right): Zhen Ming, M.D., Hugh Criswell, Ph.D.,
Katie Kelm, and George Breese, Ph.D.
Early hypotheses about the mechanisms by which alcohol increases GABAergic function were based on findings that GABA receptor antagonists and inverse agonists blocked the effects of ethanol. These compounds also blockthe effects of GABA by binding to its receptors. The binding occurs because of the structural similarity of the drugs (or GABA) and the receptor: the drugs are a “key” that fits into the “lock” that is the receptor site. Binding of the drug to the receptor initiates a cascade of events that results in changes in the electrical activity of the cell. Alcohol may bind directly to GABA receptors to influence nerve cell activity. However, experiments to test this hypothesis yield contradictory results and haveprompted Criswell to investigate other mechanisms by which alcohol might enhance GABAergic neurotransmission.
Based on previous work, Criswell hypothesized that alcohol’s GABAmimetic effects might be caused by alcohol-mediated increase in release of GABA from nerve cells. Nerve cells store neurotransmitters such as GABA in packets. When a nerve cell is activated, the packets of neurotransmitters fuse with the nerve cell membrane and empty their contents into the synapse—that is, the microscopic space between nerve cells. The neurotransmitter from the presynaptic neuron diffuses through the synaptic fluid to bind to receptors on the surfaces of other nerve cells in close proximity (i.e., postsynaptic neurons). The binding of neurotransmitter to receptors can activate or inhibit the postsynaptic nerve cells. Binding of GABA to receptors on a postsynaptic nerve cell generally causes inhibition of the postsynaptic nerve cell.
Criswell studies nerve cell inhibition and activation at the synaptic level by measuring electrical currents generated in individual nerve cells isolated from brains of rats. To test his hypothesis that alcohol’s GABAmimetic effects might be caused by an alcohol-mediated increase in GABA release from nerve cells, Criswell measured miniature inhibitory postsynaptic currents (mIPSCs) from isolated nerve cells to which alcohol had been applied. The mIPSC corresponds to the amount of electrical current (in this case, inhibitory current) generated in the postsynaptic nerve cell by the presynaptic nerve cell’s release of one packet of GABA. With Research Associate Dr. Zhen Ming, Criswell found that alcohol increased the frequency of mIPSCs in cells from a brain region known as the cerebellum. The additional finding that the alcohol-associated increase in the frequency of mIPSCs was prevented by pretreatment of the cells with bicuculline, a drug that blocks the postsynaptic GABAA receptor, confirms that the mIPSCs were mediated by GABA. Together, the results suggest that alcohol might enhance GABAergic neurotransmission by increasing the amount of GABA released from presynaptic nerve cells. Thus, alcohol can affect a mechanism related to GABA function without binding directly to the postsynaptic GABA receptor. Criswell and Ming found that alcohol potentiates mIPSCs in cells from the cerebellum but not in cells from the cortex. The latter result is consistent with a regionally specific effect of alcohol in the brain.
With graduate student Katie Kelm, Criswell next sought to determine the mechanism of the alcohol-associated increase in GABA release from cerebellar cells. Criswell and Kelm knew that alcohol can increase cellular concentrations of calcium and that increases in intracellular calcium mediate release of neurotransmitters including GABA. They hypothesized that increases in intracellular calcium might be important in mediating alcohol-associated GABA release from cerebellar cells. Intracellular calcium can be increased in two ways: by entry of calcium into the cell from the extracellular fluid through membrane-spanning calcium channels or by release from stores inside the nerve cell. Criswell and Kelm measured miniature inhibitory postsynaptic current from cerebellar cells to investigate the contributions of these two mechanisms to alcohol-associated release of GABA. They found that alcohol could increase miniature inhibitory postsynaptic currents even when entry of calcium into the cells was prevented by bathing cells in a calcium-free solution or by blocking the membrane-spanning calcium channel. These findings suggest that extracellular calcium is not necessary for alcohol-associated release of GABA from nerve cells. They also found that manipulations that depleted intracellular calcium stores significantly reduced the ability of alcohol to cause release of GABA from nerve cells. This research, published this year in The Journal of Pharmacology and Experimental Therapeutics, constitutes the first demonstration that calcium release from internal stores appears to be necessary for alcohol-associated GABA release from nerve cells. Kelm was awarded the prestigious Enoch Gordis Student Research Recognition Award for their work at the 2007 Research Society on Alcoholism meeting.
A large body of evidence has also shown that ethanol increases the concentrations of endogenous GABAergic neuroactive steroids. Neuroactive steroids can also bind to GABA receptors to affect GABAergic neurotransmission. Criswell has begun to investigate the possibility that alcohol enhances GABAergic neurotransmission through increases in neurosteroid availability at the synapse. His initial work focuses on the neurosteroid allopregnanolone—one of the most potent known modulators of GABA function. Allopregnanolone is made from progesterone in the adrenals and brain in the presence of the enzymes known as 5-alpha-reductase and 3-alpha-hydroxysteroid-dehydrogenase. Ingestion of alcohol increases progesterone and allopregnanolone in specific areas of the brain. Possibly, alcohol-associated increases in progesterone and allopregnanolone constitute another mechanism by which alcohol enhances GABAergic neurotransmission. Consistent with this possibility, the allopregnanolone precursor progesterone potentiated the electrical effects of GABA when applied to nerve cells in brain slices in recent experiments by Criswell and Ming. This effect was observed in brain areas where the enzymes responsible for converting progesterone to allopregnanolone are present, but not in brain areas that do not contain the enzymes. This work suggests that allopregnanolone may be synthesized from progesterone in brain and contribute to the GABAergic effects of ethanol.
Figure: Two mechanisms by which alcohol affects GABAergic transmission. Alcohol increases GABA release from presynaptic nerve terminals. Alcohol also increases neurosteroid levels in the synapse that can act at GABA-A receptors.
Criswell and his colleagues have elucidated two ways in which alcohol can affect GABAergic neurotransmission without binding directly to GABA receptors: by increasing GABA release and, possibly, by increasing the availability of neurosteroids that act at GABA receptors (Figure). Criswell is not stopping there. “There are many other avenues to explore,” says Criswell. “Just yesterday, people from the lab down the hall suggested that we assess the effects of alcohol byproducts such as acetate in our model. A few months ago, we worked with another laboratory in the Center to apply our model to the study of mice with genetically altered GABAergic systems. We have a wealth of people who are idea generators here, and we have the technical expertise to test these ideas thoroughly through collaborative work. That’s what is so exciting about doing science at the Bowles Center.”