VOLUME 12, NUMBER 15 • FEBRUARY 16, 2001

Opioid Gene Knockout Mice Show Greater Fear and Anxiety

Donald Pfaff is head of the Laboratory of Neurobiology and Behavior, where researchers are studying fear and anxiety response in mice.

Like many other animals, rodents tend to "freeze" when they sense danger. A new paper from Professor Donald Pfaff’s Laboratory of Neurobiology and Behavior, published in the Proceedings of the National Academy of Sciences, helps explain why.

Results of experiments on a new breed of genetically altered mice indicate that opium-like substances naturally found in the body, particularly one called enkephalin, inhibit fear and anxiety. Previous research findings had implicated these internal opioid substances in the regulation of female sexual receptivity, the ingestion of sweet solutions, and fear-related behaviors.

The Pfaff lab, along with colleagues at the Robert Wood Johnson Medical School and Queens College, reports that mice missing the gene for enkephalin had heightened reactions to three different fear- and anxiety-producing situations. The new paper strongly suggests that opioids, and particularly enkephalin gene products, act naturally to inhibit fear and anxiety. Without them, mice exhibit an exaggerated response to fearful situations.

"This mouse may comprise a new genetic model of chronic fear and anxiety," says Andre Ragnauth, a postdoctoral associate in the Pfaff lab and first author on the PNAS paper.

The preproenkephalin knockout (PPEKO) mice were tested in three different types of experiments: fear conditioning (which measures an animal’s learned fear response), open field activity and dark-light transition. In each case, the enkephalin knockout mice showed exaggerated responses to fear- or anxiety-provoking environments, compared to their wild-type and heterozygous control groups.

In one test, the mice were put into an "open field" experiment, which creates an anxiety-provoking situation. A mouse that is anxious when exposed to unfamiliar situations will move around less in the new environment, and when it does move, it will tend to stay by the walls. In contrast, an animal that is not anxious will spend more time exploring the center of the open field and will travel more distance. PPEKO mice traveled significantly less distance in the center squares of the open field and spent significantly less time in the open field than did the corresponding control groups.

The chart shows alterations in fear conditioning over two days in wild-type mice (WT), PPEKO mice and heterozygous mice (HZ). Researchers measured the amount of time the animals "froze" during a pre-shock auditory stimulus and to a mild shock on the foot.

In a second type of experiment, the mice were placed in a test chamber that had both lighted and dark areas. This test asks two questions: If a mouse is placed in the dark side of a standard test chamber, will it come into the light? And if so, how much time will it spend there? An animal that is afraid will tend to stay in the darkness. The lab’s findings indicated that the PPEKO mice were more fearful: These animals spent significantly less time in the light side than did either the wild type or heterozygous controls. They also tended to be less active once they were on the light side of the compartment. (This result was not simply because they moved less in general; PPEKO mice on the dark side of the chamber moved as frequently as the control groups).

In an "open field" experiment, PPEKO mice covered significantly less distance than the control groups (WT and HZ), and they spent less time in the center of the open field. The upper panel (A) represents alterations in the amount of distance traversed by the three types of mice, and the lower panel (B) represents the amount of time spent in the center.

The top figure shows the amount of time the three strains of mice spent in the "light" compartment of a light-dark experiment. The middle panel shows the level of activity while in the light. The lower panel shows activity levels while in the dark.

In a third type of experiment, animals of all three genotypes were evaluated to measure their learned response to fearful situations. Each of the mice was placed in a test chamber and allowed to explore freely for two minutes. Following this, the animal was exposed to a white noise-conditioned stimulus ending with a mild electric shock to the foot. Then, a minute and a half later, the mouse was returned to its home cage. The next day, the mouse was placed back in the testing chamber and again exposed to identical conditions. The researchers measured animals’ freezing behavior both upon hearing the auditory stimulus and upon experiencing the foot shock. All three genotypes froze in response to the auditory stimulis on the second day (as compared to the first day). Likewise, their post-shock "freezing" was significantly greater on day two than on day one. The PPEKO mice, however, froze for significantly more time on day two than did the other mice. PPEKO mice also displayed a significant increase in freezing after the shock on day two (both relative to the level on day one and to the control groups).

Thus, in each experiment, the enkephalin knockout mice appeared to show an exaggerated response to an anxiety-provoking situation.

Interestingly, when the PPEKO animals were tested for sugar intake and sexual responsiveness (which also are related to endogenous opioid systems), their responses were not significantly different from those of the control groups. "We were surprised that the enkephalin knockout animals failed to display either substantial decreases in sucrose consumption or reductions in lordosis [sexual responsiveness] behavior," says Ragnauth. "The stable ingestive and sexual behaviors argue strongly that the increased fear and anxiety (as evidenced by reduced activity and movement in light and open areas and ‘freezing’ in threatening situations) were due to a selective deficit and not to some generalized and non-specific debilitation."

Future work will concentrate on the genes for opioid peptide receptors.

Ragnauth’s and Pfaff’s coauthors are Alwin Schuller of the Robert Wood Johnson Medical School, Postdoctoral Fellow Maria Morgan of the Pfaff lab, Research Assistant Johnny Chan of the Pfaff lab, Assistant Professor Sonoko Ogawa of the Pfaff lab, John Pintar of the Robert Wood Johnson Medical School and Richard Bodnar of Queens College, City University of New York.

This research was supported by the National Institutes of Health.