Yet, scientists have had a hard time putting a finger on the precise nature of these imbalances. What, exactly, goes wrong?
Two Harvard Medical School investigators, Robert Greene , associate professor of psychiatry, and Donald Rainnie , instructor of psychology in the Department of Psychiatry, have recently put together an understanding of at least one way a genetic change can wreak chemical havoc in the brain. In experiments with mice, they have shown that a genetic mutation-resulting in unusually aggressive, fearless behavior-may work its devious effects by disrupting production of an important brain chemical, serotonin.
This research, reported in the Oct. 14 issue of Science, could help bring about a better understanding of human psychiatric disorders.
"You see a behavior [in the mutant mice] that is consistent with what people talk about as personality traits. And these are the same personality traits that, when they go awry, seem to form the basis of some psychiatric disorders," says Greene, a research physiologist at the Brockton Veterans Administration Medical Center.
The mice studied by Greene and Rainnie were deficient in an important enzyme called alpha-calcium-calmodulin- dependent kinase II (CAMKII). Susumu Tonnegawa, a Nobel Prize-winning biologist at Massachusetts Institute of Technology, developed the mutant mice using a "gene- knockout" technique that selectively silences individual genes.
In normal mice, CAMKII accounts for about 1 percent of total brain protein-a significant proportion considering the hundreds of chemical players in the brain. Not surprisingly, MIT biologist Chong Chen quickly found that mice missing both copies of the CAMKII gene (homozygote mice) suffered from severe behavioral and cognitive abnormalities.
"The homozygote you tend to think is going to be a little screwed up because it's got 1 percent of its total brain protein missing," says Rainnie.
But Chen found that mice with one good copy of the gene (heterozygotes) also behaved in odd ways. Whereas normal mice when presented with a shock characteristically assume an immobile crouching posture (a typical fear response known as freezing), the heterozygotes did so only a small percentage of the time.
Similarly, when placed in the center of an open field, the mutant mice would dawdle, whereas normal mice immediately sprint to the perimeter (a fear response known as thigmotaxis).
"Now if you're stupid enough to stay in the middle of the field when you know there's a hawk up there, you're not going to last very long," says Rainnie. "So you don't see this [gene] deletion very often in natural populations-and it wouldn't do to try and fight the raptor."
But the mutants-having been developed in the lab instead of nature-displayed little neck-saving behavior. When intruding upon another mouse's territory, the heterozygotes continued to fight in the face of the resident's attack, while the normal lab mice, benefiting from millions of years of untampered evolution, instinctively froze or fled.
In short, the heterozygotes were both unusually aggressive and fearless. "So the interesting thing for us was that the heterozygote showed a very clear phenotypic [behavioral] difference from the wild [or normal] type," says Rainnie.
He and Greene, in their research, sought to identify the chemical pathway that led to this aberrant behavior. They began with two clues. First, it was widely thought that the neurotransmitter serotonin might be involved in regulating fear and aggression. Second, serotonin was believed to be the end product of a conversion process involving CAMKII-the deficient enzyme. As such, it seemed likely that some defect in serotonin transmission might account for the mutants' abnormal behavior.
To determine if this was so, the two researchers-using microelectrode recordings-compared serotonin-producing brain regions in the mutant mice with similar regions in normal mice. This work pointed to a cluster of serotonin-producing neurons in a region of the midbrain, known as the dorsal raphe nucleus, as an area that might be most affected by the gene deletion.
This relatively tiny cluster of neurons has vast projections throughout the nervous system, Greene notes. "So [this region] modifies the way in which the rest of the nervous system is functioning."
He and Rainnie also knew that the dorsal raphe nucleus has low levels of CAMKII. Any cutting back of this enzyme, such as might occur by knocking out a single gene (as in the heterozygote), could have a significant effect on this brain region.
But what was causing the defect in serotonin transmission in this cluster of neurons? Normally, serotonin is transmitted in the dorsal raphe nucleus by a three-step process: Serotonin is released by one neuron and then activates receptor molecules on the receiving (or postsynaptic) neuron. A third step is removal of serotonin from the synapse (the cleft between the two neurons).
In order to tease apart these three processes-release, receptor activation and re-uptake-Greene and Rainnie had to probe the electrical activity of individual neurons, which can be kept alive for as long as seven hours in a bath of artificial cerebrospinal fluid.
"The cells don't know they're out of the brain. They're living and reacting exactly as when they're in the brain," says Greene.
He and Rainnie immersed the cells in serotonin. If the postsynaptic receptors were faulty, there would be little or no response to serotonin, and, hence, little or no electrical activity. But electrical response in the mutant neurons was normal. "That implies that the receptors on the postsynaptic cell bodies were fully funtioning," says Rainnie.
Disentangling the remaining suspects-serotonin release and re-uptake-was more complicated. Normally, the two processes go hand in hand: Serotonin is removed from the synaptic cleft almost as soon as it is released. If this were not the case, serotonin would feed back from the cleft onto the presynaptic neurons, thereby inhibiting their activity. "They're basically saying, 'We're firing too much, we better slow down,'" says Rainnie.
In order to find the true culprit, the researchers decided to tie the hands of one and see how the other reacted. They blocked serotonin re-uptake by bathing the cells in Prozac, which specifically inhibits the reabsorption of serotonin from the synaptic cleft.
If release was proceeding normally in the mutants, serotonin should start accumulating in the synaptic cleft. This buildup would signal the presynaptic neuron to cease firing. But if serotonin was being released at an abnormally low rate-due to a genetic mutation-then this chemical would accumulate much more slowly in the synaptic cleft, or not at all in the case of the homozygote. Thus, the feedback "shutdown" effect would be less dramatic.
They found that Prozac caused only a gradual muting of electrical activity in the heterozygote cells, and nearly no change at all in the homozygotes. The CAMKII deficiency in the mutants could thus be linked to a dampening in the production of serotonin.
Whether this dampening might actually be causing the mutants' abnormal behavior-the low levels of fear and high levels of aggression-remains to be shown. "It's guilty by association," Rainnie says. Adds Greene: "We think it's very guilty."
Their research may lead to a fuller understanding of the biological predisposition to certain psychiatric disorders, such as borderline personality disorder. Disorders that involve such behaviors as increased risk taking or inappropriate, intense anger may involve genetic pathways similar to the one identified by Greene and Rainnie in mice.
However, they caution that many such disorders are due to the convergence of more than one genetic disruption. Says Rainnie: "It's a truism that there is no one receptor, no one transmitter in the brain that regulates individual responses and individual behavioral patterns. It's an amalgam. It's a concert, really, of the transmitters that are out there."
--Misia Landau
Copyright 1994, President and Fellows of Harvard College. Multiple distribution or commercial reproduction by permission only.
