Monoamine oxidase (MAO) is an enzyme that comes in two flavors: A and B.

MAO-A preferentially metabolizes the neurotransmitters serotonin, norepinephrine and dopamine. In research, the MAO-A knockout mouse is a model for the molecular basis of aggression. Pharmaceutically speaking, MAO-A inhibitors are used in the treatment of depression, though second-generation antidepressants such as tricyclics or selective serotonin reuptake inhibitors (SSRIs), now are more frequently prescribed.

In contrast, the MAO-B knockout mouse has no aggressive phenotype, but it is resistant to Parkinson's-inducing agents, and thus, MAO-B inhibitors are used to treat the symptoms of Parkinson's disease. MAO-B's preferred substrate is phenylethylamine (PEA), which is present in the brain in low concentrations and thought to act as a neuromodulator.

Given the disparate effects of manipulating the MAO-A vs. MAO-B systems, the MAO-A/B double knockout mouse poses interesting opportunities for researchers and its origins already are a lesson in the vagaries of experimental research.

Here's how Jean Shih, professor of molecular pharmacology and toxicology at the University of Southern California, described its genesis to BioWorld Today: "We were trying to make a double knockout, but you cannot do it by breeding because the genes are so close together - pretty much right next to each other on the X chromosome. So we had to use a molecular genetics approach, which is a lot more work. And while we were working on it, we noticed this extremely nervous mouse in the MAO-B knockout colony.

"At first it was just annoying to the technician. Every time she opened the cage it jumped out on her, so she asked whether she could sacrifice it. I thought, She has to deal with this,' and said go ahead.

"Then after a few months we got a second one, and that's when we started joking that Maybe this is it. Wouldn't that be great, we wouldn't have to do all this work.' So we tested its urine, and there was no 5-HIAA" - the major metabolic product of MAO-A's oxidation of serotonin.

Shih also is senior author of a paper describing the double knockout's characteristics in the Sept. 17, 2004, issue of the Journal of Biological Chemistry. The paper is titled "A Spontaneous Point Mutation Produces Monoamine Oxidase A/B Knock-out Mice with Greatly Elevated Monoamines and Anxiety-like Behavior" and was co-authored by researchers from the University of Southern California and the Veteran's Affairs Greater Los Angeles Healthcare System.

The researchers first confirmed that the mouse was indeed a double knockout through direct measurements of MAO-A transcripts and protein. Both were absent from the putative double knockouts. The knockout of MAO-A was determined to be due to a point mutation that introduced an early stop codon. They then set out to determine the effect of the double knockout vs. single knockouts, regarding neurotransmitter levels and behavior.

Neurotransmitter levels revealed that there apparently is redundancy between the work of MAO-A and B under normal circumstances. Though it is mainly MAO-A that oxidizes serotonin, norepinephrine and dopamine when both enzymes are present, in the double knockout those neurotransmitters "increased much more substantially than in the [MAO-A] single knockout," Shih said. "This tells us that B is oxidizing these things when A is not there." Serotonin, for example, is usually increased by about twofold in the MAO-A knockout mouse, but was increased 8.5-fold in the double knockout.

Given the very different behavioral consequences that are observed in MAO-A vs. MAO-B knockout mice, the researchers were interested in determining the behavioral effects of knocking out both enzymes.

"They were very stressed out mice," Shih said. Hyper-reactivity - the jumpiness that doomed what was presumably the first double knockout in the colony - is a general feature of the animals.

When researchers tested for anxiety in a so-called elevated-plus maze, the animal's behavior indicated that they were more anxious than their wild-type counterparts. In their experiments, the researchers did not directly test the double knockouts against single knockouts of either MAO-A or MAO-B, but previous experiments have shown that neither type of single knockout differs from wild-type animals in its behavior in the elevated-plus maze, suggesting the single and the double knockouts might have different behavioral phenotypes.

In fact, the animals could be described as too stressed to be aggressive. The scientists also tested the double knockouts in the so-called resident-intruder paradigm, where a singly housed mouse is confronted with another mouse in its cage. The double knockouts did not take kindly to such visits. Whereas wild-type mice attacked an intruder after an average of just under nine minutes, double knockouts on the average took all of 15 seconds to go after their unwelcome guests.

However, while the double knockouts showed a clear increase in nonsocial behavior and were quicker to attack, their overall aggression scores were no higher than those of wild-type mice. In the paper, the researchers attribute that to the fact that "classical attack sequences did not as clearly establish themselves in the mutant. . . because of the hyper-reactivity of the animals and the extensive pursuit."

Shih pointed out that the spontaneous generation of two double knockouts in the MAO-B knockout colony in the space of a few months might itself provide a clue to another property of PEA, whose role in the brain is less clear than that of the classic neurotransmitters metabolized by MAO-A.

"It may be mutagenic," she said.

She also said that the combination of transmitter level and behavioral results has convinced her that "looking at increases or decreases in neurotransmitter is not enough. Looking at the extent of the increase is important." Based on her findings, she hypothesized that "at different degrees of increases, serotonin may induce different signaling pathways that finally lead to different behaviors."

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