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Zapping Brain Cells: 1: Fetal alcohol syndrome

POSTED 30 NOVEMBER 2006

Fetal alcohol: Play the blame game
The first proof that alcohol killed cells in the developing embryo came around 1981, when Kathleen Sulik of the University of North Carolina looked at cells that form the face, limbs and part of the nervous system in young embryos. Since these structures were known victims of fetal alcohol syndrome, Woman in brilliant blue sits next to large microscope in green tiled roomSulik concluded that cell death was causing some of alcohol's damage to the fetus.

Sulik started working long before apoptosis was recognized as a cell-killer. After apoptosis was identified in the early 1990s, the mystery of fetal alcohol syndrome began to unravel. In 1998, Martina Cartwright, a graduate student, and Susan Smith, professor of nutritional science at the University of Wisconsin-Madison, found that chemically blocking the cell-suicide program protected chicken brain cells from ethanol treatment (see "Ethanol-induced neural ..." in the bibliography).

Susan Smith, of the University of Wisconsin-Madison, was the first to show that alcohol causes programmed cell death in embryos. She's in front of a microscope used to detect calcium, an apoptosis promoter, inside cells.

This was the first direct implication of apoptosis in fetal alcohol syndrome. The next year, Marieta Heaton of the University of Florida saw a similar result among mice that made an abnormal amount of protein that was known to suppress apoptosis. Her results further fingered cellular suicide in fetal alcohol syndrome.

Exploring the mechanics of cell suicide
John Olney, a professor of psychiatry at Washington University, came at the issue from a different direction: brain injury. While studying how neurons die after brain injury, he blamed some of the deaths on over-stimulation by glutamate, a neurotransmitter (a compound that carries signals between neurons). Logically, blocking glutamate should protect neurons from dying from over-excitation. It did.

But blocking glutamate also increased apoptosis. "That was a puzzling observation," Olney says.

Apoptosis, as mentioned, is a crafty suicide, and the dead neurons depart the scene of the crime. To detect apoptosis in rodent brains, Olney looked for activated caspase-3, which is called the "executioner enzyme" because it forms when cells are doomed to suicide. He also looked for silver, which enters neurons during apoptosis and is easily seen under the microscope. This kind of research, obviously, can only be done on lab animals, but you must look quickly: fragments of apoptized cells disappear within hours.

Two slices of brain, one with a few black dots sprinkled throughout, the other with many
The enzyme caspase-3 also detects apoptosis. Here, we see sections of normal mouse brain (left) and a mouse brain eight hours after a single ethanol treatment (right). Antibodies to caspase-3 show neurons that are committing suicide.
LEGEND: PC = parietal cortex, Cing = cingulate cortex, HC = rostral hippocampus
Courtesy John Olney (see "Fetal alcohol syndrome at the cellular level ..." in the bibliography)

Aware that blocking glutamate receptors causes programmed cell death, Olney began to check other chemicals that affect the brain. When he came to agents that promote GABA (the major inhibitory, or calming, neurotransmitter), apoptosis arose again. "We tested a large number of those agents, and every one triggered apoptosis in developing brains."

A cell transforms from sphere to globby messWhen a cell undergoes apoptosis, white blood cells gobble the garbage. That's why the suicide leaves no lasting sign. Image: NIH

Under the microscope, Olney found that ethanol, the active ingredient in wine, beer and booze, decimated neurons in lab animals that received alcohol during the making-connections stage of neural development. Olney's 2000 report on this added to the growing evidence that ethanol triggers apoptosis in the brain: "Transient ethanol exposure can delete millions of neurons from the developing brain. This can explain the reduced brain mass and neurobehavioral disturbances associated with human fetal alcohol syndrome." (see "Ethanol-Induced Apoptotic..." in the bibliography.)

Just one dose of alcohol was enough to kill neurons by the thousands -- even millions.

Murder, he wrote
Olney had observed that drugs that block glutamate's stimulation of neurons could cause programmed cell death. Then he saw the same effect with chemicals that increase the calming action of GABA. He concluded that when neurons were making connections, it was deadly to block the excitation -- or to increase the calming. "We found that interfering with two types of neurotransmitters could trigger neural apoptosis in the developing brain," he says. "What did they have in common? Both interferences result in slowing neuronal activity."

Nerve cells that are befogged by these bogus messages are out of luck, he adds. "We were putting the cells asleep, or out of synchrony with other cells that were continuing to operate at the normal activity level," which "triggers a message: 'Kill yourself, because you are not making synaptic contact with the appropriate timing and sequence.'"

The relevance to fetal alcohol syndrome is this: Ethanol, the drug of choice for millions, slows neurons with both mechanisms. Alcohol both lowers glutamate excitation and raises GABA inhibition. No wonder it can kill developing brain cells.

Two slices of brain, one with lots of working tissue, the other black with death
Silver is incorporated in neurons that are committing suicide. Here, silver staining contrasts a normal (A) and alcohol-treated mouse brain (B). 24 hours after ethanol treatment, a few neurons are dying in the control brain (notice the black specks), while vast numbers are dying in the ethanol-treated brain. Silver staining can catch apoptosis in the act, but only if the animal can be sacrificed and placed under the microscope. Photo: Courtesy John Olney (see "Fetal alcohol syndrome at the cellular level ..." in the bibliography)

The basics on booze
To explain why a drug that humans have been guzzling for thousands of years causes such massive cell death, Olney stresses that during normal brain development, nerve cells are connecting with each other at the synapses. The human brain may be the most complicated object in the universe, and the timing and pattern of its connections needs precise orchestration. Otherwise, the brain can never expect to appreciate cheese (mouse) or cheesy movies (human).

Data from Fetal Alcohol Spectrum Disorders Center for Excellence

"Nerve cells have to be making synaptic contacts on a very strict time schedule, and in a very particular sequence," Olney says. "Every nerve cell is making thousands of synaptic contacts, and each one is a specialized contact that involves one kind of transmitter or another. It's enormously complex, and it all happens in a specific sequence. ... All nerve cells are programmed to commit suicide if they don't integrate."

Timing matters in birth defects, Smith stresses. For every agent that harms the unborn, "there is a critical window of sensitivity." The horrendous birth defects caused by the sleeping pill thalidomide, for example, "only occurred if it was given during the stage of limb outgrowth. After that, the limb is deaf to thalidomide."

In normal brain development, cellular suicide is healthy because disconnected neurons are useless. Like cleaning the attic, apoptosis clears clutter. Unfortunately, alcohol changes the equation by preventing normal synaptic connections from forming on schedule. The result is a legion of misfit neurons that must commit suicide for the good of the organism.

In other words, cell death in fetal alcohol syndrome results from a natural act gone awry.

Alcohol becomes the Jack Kevorkian of the developing brain.

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Megan Anderson, project assistant; Terry Devitt, editor; S.V. Medaris, designer/illustrator; David Tenenbaum, feature writer; Amy Toburen, content development executive

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