Online Issues

ILAR Journal Vol 45(1)

Introduction

Thinking Outside the Mouse Box: The Importance of Comparative Laboratory Animal Models in Research

Emilie F. Rissman

Emilie F. Rissman, Ph.D., is a Professor of Biochemistry and Molecular Genetics at the University of Virginia School of Medicine, Charlottesville, Virginia.

We live and work in an era in which we introduce genes into new host organisms in a matter of days, sequence genes in hours, and identify homologies between genes in minutes. The power of genetics has revolutionized basic research science and drug discovery, and it is beginning to affect patient care. This movement has also advanced the attractiveness of working with genetic model organisms.

For the laboratory animal community, the model organism of choice has become the laboratory mouse. The genome of one mouse strain, C57BL/6J, has been almost fully sequenced. The availability and use of knockout and transgenic mice are widespread, and it is now possible even to hire biotechnical companies to produce designer mice. The next generation of mice with temporal and/or tissue-specific control of particular genes is on its way and will soon replace the earlier models of knockouts and knockins. And so the case can be made that there is no reason to continue research programs in rats, dogs, cats, or nonhuman primates, let alone "exotic" mammalian and nonmammalian vertebrates. The "supermouse" breeds faster, has a shorter generation time, produces more offspring, and can be inserted into any research program in place of any other animal. However, if you agree with the preceding statement, I urge you to delay final judgment until you have read the articles in this issue.

Indeed, laboratory mice are fabulous animals with many features, beyond genetics, that make them superb models for studies of physiology and behavior. In my own laboratory, colleagues and I have been engaged in an active mouse research program since 1996. However, we have not discarded the comparative approach that formed the foundation of knowledge for many of us because we know that some problems require comparative solutions.

For this issue of ILAR Journal, I have solicited contributions from just a few of the many research laboratories in the United States that utilize the comparative animal model approach. In each of the articles that follow, the respective model possesses a unique feature that makes it ideally suited for studying a particular problem. All of the articles in the issue focus on topics in reproduction and behavior, yet because evolution acts via differential reproduction, this "trait" exhibits enormous variability both between and within species. Variation between and within species is important and cannot be ignored. Discovery of variability in physiological processes and their underlying molecular and genetic mechanisms can reveal creative "solutions" to basic biological, as well as applied clinical, problems.

In the first article in this issue, Paul Heideman (2004) presents a strong case for the study of genetic variation, and for using models that exhibit this feature. Laboratory mice, particularly the most commonly used inbred strains (e.g., C57BL/6, DBA, BALB/C), were purposefully produced by generations of brother-sister pairing to create homogenous lines. Genetic variability was eradicated to simplify identification of mutations. However, as Dr. Heideman effectively describes, in human populations the variation in complex physiological pathways that has been produced by environmental and genetic interactions has important implications for medical treatment. One complex physiological response exhibited by a large number of vertebrates is photoperiodism--the ability to synchronize fertility to the times of the year when resources are most abundant and when offspring are thus most likely to survive. Resource availability is signaled by the predictable seasonal changes in daylength. This pathway involving the hypothalamic-pituitary-gonadal-pineal axis is intertwined with regulation of reproduction on all levels, from the production of gametes to the display of appropriate mating behaviors. Laboratory mice are derived from stock that at some point in their life history displayed this trait, but now they are well adapted, in fact selected, for year-round breeding under laboratory conditions, and they have lost their photoperiodic response. Wild mice like Peromyscus, however, are quite sensitive to photoperiod. They regress their gonads when daylengths are shortened in the fall, and they regrow them during spring. Moreover, there is natural variability in this trait (and likely this set of genes) in laboratory populations that are only a few generations removed from the field.

In the second article in this issue, Theresa Lee (2004) addresses an aspect of reproductive physiology that is closely related to photoperiod, circadian rhythmicity. The ability of organisms to display daily rhythms is ubiquitous in plants, bacteria, and animals. As many scientists have documented, the consequences of disruptions in human circadian rhythms range from personal discomfort in the form of jet lag to national catastrophes such as Chernobyl, where fatigue may have been a major contributing factor (Åkerstedt 2003; Buijs et al. 2003; Hastings 1998). Although hamsters, rats, and mice have been used as models to study the formal and biological properties of these rhythms, all of these animals are relatively asocial and strictly nocturnal. In contrast, humans are gregarious and active primarily during the day. Thus, Dr. Lee and her colleagues have pioneered use of diurnal mammals to study circadian rhythms. Octodon degus is a medium-sized social rodent that has robust diurnal circadian rhythms; and like humans', these rhythms are sensitive not only to the daily light:dark cycle but also to social influences. In her article, Dr. Lee presents the formal characterization of diurnal circadian rhythms and some of the preliminary neurobiology. This developing story will be extremely useful to help us understand how human biological timing is regulated.

In the third article, by Jennifer Temple (2004), the animal model is the musk shrew, a lovely creature similar to the size of mice but evolutionarily closer to primates than rodents. The interaction between nutrition and reproduction can be studied in any organism because low nutritional status invariably leads to infertility. Yet the selection of the musk shrew has many advantages, some of which Dr. Temple knew when she began her studies and others that she discovered in the course of her investigations. Because shrews store less body fat and have higher metabolic rates than mice or rats, they are more susceptible to small variations in food intake. Most of the work on this topic has utilized hamsters and rats and has revealed effectively that many aspects of the reproductive cascade are down-regulated when animals ingest significantly less food over several days. Although sedentary rodents fed an endless supply of food without access to any exercise might be an appropriate model for the "couch potato," most of the human population does not have the luxury to live under these conditions, and the musk shrew is a more appropriate model to investigate the basic biology underlying nutrition/reproduction interactions. Dr. Temple's work has revealed for the first time that one neurotransmitter, an isoform of gonadotropin-releasing hormone, may act as part of a gating mechanism that links nutritional and reproductive status. The function of this hormone had been a complete mystery until this discovery, and the isoform present in human (and nonhuman primate) brain is absent in rodents. Thus, work in this "exotic" species will provide critical insights into the role of this hormone in human reproduction.

In the fourth article, Brandon Aragona and Zuoxin Wang (Aragona and Wang 2004) describe the prairie vole (Microtus orchrogaster, a close relative of Mus musculus), which is used as a model species to study neural and hormonal regulation of social behavior. Voles, also called field mice, are widespread in nature, and different species exhibit a large degree of flexibility in their social structure ranging from asocial to highly gregarious. Prairie voles are on the extreme end of the scale, and they live in multigenerational family groups in the wild. Before the administration of DNA paternity tests, researchers thought these mammals mated in a monogamous fashion. However, we now know that ex-pair matings can and do occur. Yet when given a choice, males (and females) always prefer to associate with their familiar mating partners versus strangers. This preference behavior, or "pair bonding," is rare among mammals. Together with the authors of this article, the network of researchers who have made great progress in uncovering the hormonal and neural underpinnings of pair bonding includes, but is not limited to, the following: Sue Carter (1998), Larry Young and Tom Insel (Young et al. 1998), Diane Witt (1997), and Joe Lonstein and Geert De Vries (Lonstein and De Vries 2000). The potential information from such studies of affiliative behavior is extremely valuable in a violent world. In fact, genetic polymorphism, in the same genes involved in pair bonding in prairie voles, has been noted in psychiatric patients.

In the next article, Sarah Woolley, J. T. Sakata, and David Crews (Woolley et al. 2004) present a compelling example of the use of an unusual animal model to study the evolution of behavior. The parthenogenetic desert-grasslands whiptail (Cnemidophorus uniparens) is a hybrid produced by two closely related whiptailed lizards. Both of the parental strains are diploid and reproduce sexually. Often hybrids (such as mules) are infertile; however, the parthenogenetic whiptailed lizards have triploid chromosomes, all individuals are females, and their eggs enter meiosis without sperm activation. Thus like inbred mice, all individuals are theoretically clones. The Crews laboratory has developed this animal model over the past 20 yr. They have successfully perfected all of the tools (from animal husbandry to in situ hybridization) needed to conduct modern behavioral neuroscience studies with this species. One of their surprising findings is that the female parthenogenetic lizards readily perform both female- and male-typical sexual behaviors. The hormonal and neural control of both sexual behaviors within the same species has been characterized and compared with one of the ancestral species. In addition, Dr. Crews and colleagues have created males by hormonal manipulations of the developing eggs. These males are genotypically identical to their sisters. This animal provides us with a very unique model to study the ontogeny of sex differences in behavior. Such model systems also may eventually shed light on the biological bases of gender identity.

The next contribution, by Matthew Lovern, Melissa Holmes, and Juli Wade (Lovern et al. 2004), illustrates a more traditional approach to the study of sex differences in behavior. The green anolis is a common small lizard that has two sexes and displays sexual reproduction. Their courtship display involves several stereotyped behaviors; the dewlap, a piece of skin under the chin, figures prominently in these displays. Both males and females extend the dewlap while head bobbing in either an aggressive (intrasexual) or a sexual (intersexual) context. Males, however, have a larger dewlap that is controlled by a greater muscle mass, and they express head bobbing more often than females. The use of behavioral sex differences (e.g., song in male birds, mounting and thrusting during copulation in male rodents) has become the classic approach to studies of sex differences and has led to the discovery of many sex differences in neuronal organization and adult function. The standard dogma is that sex differences are the product of differences in hormone levels in neonatal male versus female brains. Once hormones act on neural circuits during development, by stimulating cell death, birth, and/or migration, the same hormones are required in adults to activate the appropriate behaviors. Because both male and female anolis display dewlap extension and head bobbing at all times of the year, it is likely that this system is less highly sexually dimorphic than some of the more traditional sexual behaviors. Human sex differences are also far less dimorphic than behaviors such as singing in canaries (males, but not females, sing). For example, sex differences in human cognitive behaviors are not distinct but rather require repeated replication and elegant statistics to be revealed. Clearly, the development of subtle, sexually dimorphic systems such as the anolis will assuredly shed light on human sex differences, which can be small but extremely important.

In the final article, Randolph Krohmer (2004) describes the reproductive biology of the male red-sided garter snake. These animals have a very broad range in North America, and at their highest latitudes, throughout winter, they live in a dormant state in very large underground groups. When they emerge in the spring, males awake first and stay at the entrance of the hibernacula ready to court as soon as females appear above ground. At the time of emergence, male testes are not producing sperm but are producing small, albeit measurable, amounts of testosterone. Courting and mating behavior in males persists after castration, thus there is no absolute requirement for high levels of circulating testosterone in this species as is noted in rodents. This feature, termed "dissociated mating," characterizes only a few species. Interestingly, nonhuman primates and humans also retain the ability to mate for years after castration in adulthood. Although these studies are still in their initial stages in the garter snake, they could have very important implications for the development of drugs to treat sexual dysfunction in men.

In sum, it is my hope that this collection of articles will provide readers with a renewed sense of the amount of diversity present in nature. It would be a shame to forego the special opportunities that these animals, and many others, provide in favor of the convenience of studying the laboratory mouse. The topics discussed herein may appear at first glance to be esoteric, but it is easy to see how the data collected in these animals can be creatively applied to problems in veterinary and human medicine.

Acknowledgment

The authors and I are indebted to Susan Vaupel for expert editorial assistance. In addition, she has kept this issue on track in the most pleasant and professional manner.

References

Åerstedt T. 2003. Shift work and disturbed sleep/wakefulness. Occup Med (Lond) 53:89-94.

Aragona BJ, Wang ZX. 2004. The prairie vole: An animal model for behavioral neuroendocrine research on pair bonding. ILAR J 45:35-45.

Buijs RM, van Eden CG, Goncharuk VD, Kalsbeek A. 2003. The biological clock tunes the organs of the body: Timing by hormones and the autonomic nervous system. J Endocrinol 177:17-26.

Carter CS. 1998. Neuroendocrine perspectives on social attachment and love. Psychoneuroendocrinology 23:779-818.

Hastings M. 1998. The brain, circadian rhythms, and clock genes. Br Med J 317:1704-1707.

Heideman PD. 2004. Top-down approaches to the study of natural variation in complex physiological pathways using the white-footed mouse (Peromyscus leucopus) as a model. ILAR J 45:4-13.

Krohmer RW. 2004. The male red-sided garter snake (Thamnophis sirtalis parietalis): Reproductive pattern and behavior. ILAR J 45:65-74.

Lee TM. 2004. Octodon degus: A diurnal, social, and long-lived rodent. ILAR J 45:14-24.

Lonstein JS, De Vries GJ. 2000. Sex differences in the parental behavior of rodents. Neurosci Biobehav Rev 24:669-686.

Lovern MB, Holmes MM, Wade J. 2004. The green anole (Anolis carolinensis): A reptilian model for laboratory studies of reproductive morphology and behavior. ILAR J 45:54-64.

Temple JL. 2004. The musk shrew (Suncus murinus): A model species for studies of nutritional regulation of reproduction. ILAR J 45:25-34.

Witt DM. 1997. Regulatory mechanisms of oxytocin-mediated sociosexual behavior. Ann N Y Acad Sci 807:287-301.

Woolley SC, Sakata JT, Crews D. 2004. Tracing the evolution of brain and behavior using two related species of whiptail lizards: Cnemidophorus uniparens and Cnemidophorus inornatus. ILAR J 45:46-53.

Young LJ, Wang Z, Insel TR. 1998. Neuroendocrine bases of monogamy. Trends Neurosci 21:71-75.