Researchers Study Mechanism of Alcohol's Effects—One Cell at a Time
I cannot live without the sparkling vintage, Cannot bear the body's burden without wine. . . I drink so much wine, its aroma Will rise from the dust when I'm under it; Should a toper come upon my dust, The fragrance from my corpse will make him roaring drunk. —From Omar Khayyam's Ruba'iyat, eleventh century A.D.As Khayyam's vivid description attests, humankind has suffered the throes of alcohol addiction for centuries. Although alcohol is one of the oldest drugs known to man, the physiological mechanisms by which it produces intoxication and dependence have yet to be elucidated.
Dr. Hugh Criswell, Research Associate Professor at the University of North Carolina's Bowles Center for Alcohol Studies, devotes his days to unraveling the mystery of how alcohol alters brain function to cause intoxication and dependence. A contemplative man, Criswell is not only a brilliant scientist, but also a musician (he plays piano and clarinet), an astronomer (he telescopically photographed the impact of the Shoemaker-Levy comet on Jupiter), and a wilderness lover (he regularly retreats to his 50 woodland acres in the North Carolina mountains). He speaks thoughtfully and thoroughly about the historical backdrop giving rise to his current research into the cellular effects of alcohol: "Alcohol was formerly believed to alter behavior by indiscriminately, globally affecting the brain," says Criswell. "Alcohol was viewed as acting non-specifically even at the cellular level, where it was said to fluidize cellular membranes. We now know that alcohol affects the brain rather selectively by altering the function of some brain regions while leaving others relatively untouched. We have also learned that alcohol acts specifically at particular sites on a given cell and fluidizes cellular membranes only at doses higher than those required to cause intoxication."
Dr. Hugh Criswell
Coming to UNC in 1988 from East Tennessee State University, Criswell began a collaboration with Dr. George Breese which continues to this day. Breese's lab had been recording the electrical activity of single nerve cells in a brain region known as the medial septum. They had found that alcohol enhanced the effects of GABA, a chemical naturally found in the brain, when applied to cells shortly before GABA was applied. GABA inhibits cellular functioning by opening a cell membrane-spanning channel and allowing negatively charged ions to enter a cell, thereby decreasing its electrical activity. Alcohol facilitated GABA's inhibition of electrical activity. These data suggested that alcohol's effects are mediated at least in part through enhancement of GABA-mediated inhibition.
Intriguingly, alcohol's enhancement of GABA-mediated inhibition of nerve cells, an effect so pronounced in the medial septum, was absent just a few micrometers away in the lateral septum. This observation led to the conclusion—ground-breaking in a time when alcohol was thought to affect the brain uniformly throughout—that alcohol affects different brain regions in different ways.
Why was alcohol affecting GABA responses in some brain regions but not others? In an attempt to answer this question, Criswell and his colleagues sought to identify the characteristics distinguishing brain regions exhibiting alcohol-mediated enhancement of GABA inhibition from those not exhibiting it. They found alcohol-responsive brain regions such as the medial septum also contained high levels of a nerve cell receptor known as the type 1 benzodiazepine receptor, whereas brain regions not responsive to alcohol contained very few type 1 benzodiazepine receptors.
The type 1 benzodiazepine receptor is a specific group of GABAA receptor proteins that surrounds a channel spanning the nerve cell membrane. When it is stimulated by drugs or by chemicals naturally occurring in the brain, the type 1 benzodiazepine receptor can cause the channel to open and allow inhibitory ions into the cell, thereby decreasing the cell's electrical activity and inhibiting its function. The observation that brain regions rich in type 1 benzodiazepine receptors were also the regions where alcohol enhanced the ability of GABA to open the membrane-spanning channel suggested that the type 1 benzodiazepine receptor might be important in determining a brain region's responsiveness to alcohol.
The Neuropharmacology Section Team, left to right: Darin Knapp, Ph.D.; Zhen Ming, M.D.; George Breese, Ph.D.; Ben Griffith; Edna Titus; Dana Rotella; Thomas McCown, Ph.D.; Sheryl Meet, Ph.D.; Hugh Criswell, Ph.D.; and Ying Qiang. Criswell more definitively pinpointed the type 1 benzodiazepine receptor as a likely substrate for alcohol in experiments employing the electrophysiological technique of microiontophoresis, in which chemicals are applied directly to individual nerve cells. Where previous work had identified brain regions, each comprised of millions of nerve cells, that both responded to alcohol and contained type 1 benzodiazepine receptors, microiontophoresis allowed Criswell to determine at the level of the individual neuron whether presence of a type 1 benzodiazepine receptor was associated with response to alcohol. Criswell spritzed both alcohol and zolpidem, a drug that selectively binds to and marks type 1 benzodiazepine receptors, onto nerve cells in a variety of brain regions prior to recording the cells' electrical activity. In 90% of cells, responses to alcohol and zolpidem were perfectly concordant: a cell that was inhibited by alcohol was inhibited by zolpidem, and a cell that did not respond to alcohol also failed to respond to zolpidem. Criswell and his colleagues had thus identified an alcohol-sensitive receptor.
Though the type 1 benzodiazepine receptor may be a structural feature important in mediating alcoholŐs effects on some cells, recent, unexpected findings have revealed that this structural feature is not always sufficient. For instance, Purkinje neurons in the cerebellum always respond to zolpidem, but often require activation of ß-adrenergic receptors or GABAB receptors before they will respond to ethanol. Criswell found that nerve cells from rats' substantia nigra reticulata, a region of the brain mediating motor function, consistently exhibited alcohol-mediated enhancement of GABA inhibition when studied in the intact animal, an effect consistent with earlier data from the alcohol-sensitive medial septum. When the substantia nigra cells were studied isolated from the animal in an in vitro preparation, however, alcohol-mediated enhancement of GABA inhibition was rarely observed. Criswell knew from previous experiments that substantia nigra cells contain the alcohol-sensitive type 1 benzodiazepine receptor and therefore should exhibit alcohol-mediated enhancement of GABA inhibition. Why the lack of effect of alcohol on dissociated nerve cells then?
Criswell and his collaborators are presently examining several hypothetical reasons why ethanol appears to enhance the action of GABA in the intact brain but loses that action when individual nerve cells are studied in vitro. The studies of cerebellar Purkinje cells described earlier raise the possibility that ethanol will enhance the action of GABA only when another input to the nerve cell, such as ß-adrenergic stimulation, is present. There are a number of auxiliary sites on the GABA receptor that may be activated by substances normally present in brain but lost when nerve cells are removed from the brain. Isolating cells from the brain environment would eliminate these endogenous factors and perhaps, prevent alcohol from having its normal effect on the GABA receptor. Dr. Criswell states that "It is becoming obvious that alcohol has a complex effect on the brain and may alter the interactions among several signaling systems. What is really exciting is that, thanks to recent progress in molecular biology and neuroscience, we now have the tools to study these complex actions of alcohol. This is a great time to be an alcohol researcher."
One Cell at a Time- Alcohol Actions - Director's Column
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