ILAR Journal Online, Volume 39(1) 1998: Animal Well-being: Immune Function, Behavior, Morphology, and Psychoneuroimmunology

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ILAR Journal V39(1) 1998
Animal Well-being: Immune Function, Behavior, Morphology, and Psychoneuroimmunology

Effects of Experience and Environment on the Developing and Mature Brain: Implications for Laboratory Animal Housing
Ann C. Benefiel and William T. Greenough

Ann C. Benefiel, B.S., is Director of the Biological Resources Facility at the Beckman Institute, Urbana, Illinois. William T. Greenough, Ph.D., is Swanlund Professor and Center for Advanced Study Professor at the Beckman Institute and in the Departments of Psychology, Psychiatry, and Cell and Structural Biology, University of Illinois at Urbana-Champaign.

INTRODUCTION

In normal animal development, experience molds the brain and behavior. The potential for experience to alter brain organization and behavioral capabilities and tendencies continues through most of, if not all, an animal's (or human's) life. A key component of this view is that a "normal" nervous system is one that has been exposed to some minimal amount of experience, allowing it to develop normally. However, laboratory animals such as rats, housed in minimally stimulating surroundings through their lives, may have abnormal, underdeveloped brains and may, under certain conditions, react in ways that are different from animals exposed to a broader array of experience.

In the context of regulations regarding current, standard animal housing and alternative housing that may be more optimal for the welfare of laboratory animals (particularly rodents), we first address special sensitivity of the developing nervous system to experience, using the term "experience-expectant information storage" (Black and Greenough 1986; Greenough and Black 1992). We then discuss lifelong information storage, termed "experience-dependent information storage" (Black and Greenough 1986; Greenough and Black 1992).

EXPERIENCE, THE BRAIN, AND BEHAVIOR

Experience-expectant Information Storage

The development of many, if not all, mammals involves certain consistencies in the information available in the typical rearing environment. For example, in visual development, it is very likely that all members of a species have been exposed to a common set of visual stimuli that, at least for diurnal animals, includes a diverse array of patterned input via the retina. Deprivation studies have indicated that the visual systems of cats and monkeys require or "expect" this stimulation for normal development. If normal pattern vision is prevented, either through occlusion (Hubel and Wiesel 1970) or through presenting a limited range of visual stimuli such as only vertically oriented lines (Hirsch and Spinelli 1970), the performance of the visual system will be relatively insensitive to stimuli at any orientation, in the case of occlusion, or at the nonexposed orientations, in the second case. Similarly, if 1 eye gets normal experience and the other is deprived, the experienced eye develops normal vision and the deprived eye becomes effectively blind (LeVay and others 1980). In both cases, there is a critical or sensitive period when normal visual experience must occur if normal vision is to develop. The changes that occur do so within the visual cortex of the brain, whereas the eyes themselves show little if any effect of these manipulations. Once this experience has occurred, the system becomes nearly invulnerable to deprivation.

The nervous system appears to have evolved a special mechanism for encoding "expected" information. During development, the visual cortex overproduces the synaptic connections through which nerve cells communicate, and the numbers peak within the sensitive period for experiential organization. Subsequently, as experience "tunes" a functional system, a subset of these connections is strengthened while the remainder is pruned away (Boothe and others 1979; Bourgeois and others 1994; Cragg 1975; LeVay and others 1980). This process is similar to that of marble sculpture, in which the functional form is achieved by removing the marble that does not belong. Hence, loss of synapses is essential to the development of a functional visual system; and survival and loss of synapses--as well as the operational quality of the system--depend on experience. Similar overproduction and loss processes have been reported for the human visual system and have been used to characterize human language and cognition development (Huttenlocher 1994).

Experience-dependent Brain Adaptation

Beyond early development, the brain continues to acquire information from experience. Much of this information is unique to the individual and hence may involve different mechanisms from those used for species-typical information storage. The nature of the environment and the animal's interaction with it determines much of the character of the brain--in fact, most of what we would use to describe ourselves to others reflects this sort of information storage.

Considerable data indicate that 1 mechanism underlying this lifelong information storage is the formation of new synapses in response to experience. The best-known laboratory example of this, and one very germane to our interest in laboratory animal welfare, is a series of experiments involving rearing and adult housing environments that differ in physical and social complexity. A typical experiment involves laboratory animals such as rats housed individually in standard laboratory cages ("individual cage" [IC¹]), animals housed in pairs in similar cages ("social condition" [SC¹]), and animals housed in a group of approximately 12 in a large cage filled with an arrangement of play and exploration objects that is changed daily ("environmental complexity" [EC¹] or "enriched condition"). It has been known since Hebb (1949) initiated this research that animals from EC housing outperform the other 2 groups on complex, appetitively-motivated tasks such as mazes. Although this may in part reflect greater familiarity with large, open, spatially-complex environments, there is also evidence that EC animals use different strategies and have more well-developed concepts of the properties of objects (Juraska and others 1984; Thinus-Blanc 1981).

Some time ago, it was found that the nerve cells of animals reared in, or as adults housed in, the EC environment have larger dendritic fields (the "input side" of the neuron) than those of the other 2 groups (Volkmar and Greenough 1972). As this finding might predict, EC rats also have more synapses per neuron (Turner and Greenough 1985). These results are compatible with the hypothesis that information arising from experience in the complex environment is encoded, at least in part, in new circuitry enabled by the added synapses. It should be noted that in addition to these brain effects, other differences include lowered body weights and slowed growth of certain organs and of bones in EC rats (Black and others 1989b). Further discussion of whether learning or some other aspect of complex environmental exposure drives and is encoded by brain changes appears below (see "Are Experience-dependent Effects Due to Learning?'').

Plasticity of Brain Support Systems (Glia and Vasculature)

The support systems of the brain--the vascular and glial systems--exhibit a form of plasticity similar to experience-dependent synaptic plasticity, although it appears to be more age-dependent and probably of shorter duration. In rats housed from weaning in EC, the capillary volume per nerve cell is about 80% greater than for IC rats (Black and others 1987). The size of the effect diminishes in rats first housed in EC as adults (Black and others 1991) and is further reduced with aging (Black and others 1989a). We compared the surface density (Sv) of astrocytic processes, identified as immunopositive for their characteristic glial fibrillary acidic protein, and found that the total surface area of astrocytes per neuron was similarly greater in EC rats reared in the complex environment from weaning than in IC or SC littermates (Sirevaag and Greenough 1991). There are indications that the astrocytic effects are largely driven by the needs of the new synapses, as glial volume correlates with synaptic numbers (Anderson and others 1994) and the glia appear to envelop synapses more thoroughly in the EC rats (Jones and Greenough 1996). Other work has suggested that oligodendroglia and the myelin with which they envelop axons are similarly sensitive to these experience manipulations (Juraska and Kopcik 1988; Szeligo and LeBlond 1977). With the synapse data, these results present a very dynamic view of the brain. Essentially all cellular components appear to adjust to the demands placed on the brain by the organism's interactions with its environment. It is clear that the anatomy and physiology of the brain vary with the housing environment. Thus, it would not be surprising for the results of some experimental designs to differ in important ways from others, depending on the housing conditions of the animal subjects.

Effects of Experience on Brain Physiology

One example of a housing-based physiological difference that is interpretable from the anatomical data comes from an experiment (T. L. Ivanco, Beckman Institute, Urbana, Illinois, personal communication, 1997) in which we recorded from the visual cortex of rats reared in EC and IC conditions while stimulating the subcortical white matter afferents to the visual cortex. In our recordings, we could identify the electrical potential from visual cortex neurons termed the "population spike," the sum of the individual firings of visual cortex nerve cells in response to the stimulus. As we expected, since the neurons in the visual cortex of the EC rats had more synapses, the summed firing reflected in the population spike was greater in this group. This result supports our anatomical work and indicates that the physiology of the brain can differ depending on the kind of housing in which the animal has been reared or maintained. While these experimental environments are extremes, relative to the laboratory norm, the results show that housing conditions can affect outcomes of experiments in ways that could make replicating work from another laboratory very difficult if the housing conditions are not also replicated.

Are Experience-dependent Effects Due to Learning?

Two basic interpretations exist for the effects of complex environmental (EC) housing on the brain. According to the first interpretation, as described above, the synaptic changes encode information from experience. In short, the synaptic changes mediate learning and memory, which occurs to a much greater extent in animals in a complex environment. In the second, contrasting interpretation, the brain is functionally much like a muscle, and its nerve cells hypertrophy when they are exercised. According to the second view, the combination of neuronal hypertrophy and neovascularization is very similar to the condition of muscles with exercise.

As a test of these alternative interpretations, we devised an experiment in which 1 group of rats underwent a great deal of learning with very little exercise and associated neural activity in the brain ("acrobat condition" [AC¹]), 2 groups had considerable exercise and little learning ("voluntary exercise'' [VX¹]in a running wheel attached to the home cage, and "forced exercise" [FX¹]on a treadmill), and another group of "cage potatoes" ("lack of exercise" [LX¹]) received neither exercise nor learning but were handled each day to control for the effects of handling. We first examined the paramedian lobule of cerebellar cortex (Black and others 1990), and we have since obtained comparable data for motor cerebral cortex (Kleim and others 1996). Results indicated that the AC rats formed new synapses compared with all 3 other groups in which synapse numbers did not differ, indicating that learning rather than neuronal activity was the important variable with regard to synapses. By contrast, the 2 exercise groups formed new blood vessels compared with the AC and LX groups, whose blood vessel measures did not differ. Thus, the formation of new synapses and capillaries is independently governed and, at least in this case, is differentially activated by aspects of particular behavioral experiences. In separate work, we found that astrocyte volume was correlated with synaptic number change both within and across groups (Anderson and others 1994). However, when training was suspended for 4 weeks, astrocyte effects decreased to nonsignificant levels, whereas synapse numbers and behavioral performance remained at presuspension levels (Kleim and others 1997). Thus, astrocytes appear to be jointly governed by the formation of new synapses and by their behaviorally maintained activity.

These findings reinforce the important roles that housing conditions can play in physiologic characteristics of laboratory animals. As we consider modifications of housing for the improved welfare of laboratory animals, we must take the potential physiologic effects of these changes into account.

Differences between Species

The foregoing findings were from studies using rats. Similar findings have been reported in mice, cats, and monkeys (for example, Beaulieu and Colonnier 1987; Floeter and Greenough 1979). However, recent studies using pigs remind us of the limits of generalization. In 2 studies (T. Grandin, Colorado State University, Fort Collins, Colorado, personal communication, 1997; Morrow-Tesch 1997), swine were reared in conditions intended to simulate the complex environmental conditions described above--basically either group housing with access to objects with which the animals could play and explore or an outside enclosure allowing social interaction and exploration. Comparison groups were housed in conditions reflecting more standard agricultural methods that provided some social contact but limited additional stimulation. Both studies found no effect on the visual cortex, which was heavily affected in the studies described above. Surprisingly, Grandin and others found somatosensory cortical dendritic branching to be greater in the animals housed under the standard agricultural conditions, in this case an indoor pen without bedding containing 2 pigs each. Morrow-Tesch, however, found no differences in somatosensory or visual cortex between preweanling pigs housed in indoor farrowing pens and those housed in expansive outdoor enclosures with straw bedding and multiple sows with piglets. Morrow-Tesch did find increased numbers of primary dendrites on layer IV neurons in primary auditory cortex of pigs housed in the noisier, inside environment from weaning to 8 weeks, leading her to implicate the differences in the auditory environment in this structural change. It is possible that the more deprived animals actually self-generated higher levels of somatosensory stimulation, particularly in the snout region, as Grandin and others suggest. Alternatively, it may be that the statement attributed to lngrid Newkirk, "... a rat is a pig is a dog is a boy," reflects the failure to recognize a basic fact known to those who study ethology and animal welfare: Different species respond to environmental circumstances in different ways.

Environmental Effects on Endocrine and Immune Systems

In this paper, we have not reviewed the data mitigating arguments that these environmental effects are due to stress responses in the subjects (for example, Uphouse 1980), except to note that rats placed in EC, SC, and lC environments do not differ in adrenal weight across studies, provided that the rats' placement occurred after about 24 days of age. (This does suggest that early individual cage housing, which caused adrenal weight increases, should be avoided.) The reader wishing to obtain additional information is referred to Black and others (1989b).

One issue regarding stress effects on the brain may be much more relevant to laboratory housing conditions. A variety of reports beginning with Landfield and others (1981) indicates that the stress history of a laboratory rat is predictive of cell loss in Ammon's horn region of the hippocampus. Other reports indicate that a possibly-related correlation between adrenal weight and astrocytic density has been seen in the dentate gyms of the hippocampal formation but not the visual cortex of the same animal, perhaps indicating a particular sensitivity of this brain region to stress (Sirevaag and others 1991). It has also been reported that very early, preweaning exposure to mild stress could protect the brain against subsequent stressful experiences (Meaney and others 1988). Recently, however, 2 reports have indicated that no Ammon's horn cell loss in aged rats could be detected, using the best modem, unbiased, stereologic techniques (Rapp and Gallagher 1996; West 1993) and hence calling into question the earlier reports of stress effects on this region. In a brief consensus discussion of the conflicting data, the authors of these studies have recently concluded that additional factors, which may include the animals' housing conditions, probably contributed to the differences across the studies (Gallagher and others 1996). A possibly-related finding is that immune function may differ with housing condition: Immune system function appears to be enhanced in EC rats relative to controls housed in standard cages (Black and others 1989b; Kingston and Hoffman-Goetz 1996). These phenomena are discussed in more detail in other papers in this issue.

IMPLICATIONS FOR LABORATORY ANIMAL HOUSING

Does Improved Neural Function Equal Enhanced Well-being?

Clearly, environment (specifically one including social contact and enrichment) has a great impact on many species' behavior, anatomy, and physiology. The 1985 amendments to the Animal Welfare Act (CFR 1992) have mandated exercise for dogs and environmental enrichment for nonhuman primates housed in research facilities. In the late 1980s, this concerned regulators, administrators, and researchers using these species as they dealt with the establishment of minimum standards and definitions of enrichment. However, publications and conference proceedings from the laboratory animal science field indicate that this legal milestone accelerated the interests of those who oversee the care of research animals in assuring animal well-being at a level beyond adequate nutrition, clean and safe housing, and freedom from unnecessary distress.

Nevertheless, interest in animal well-being has in no way simplified our understanding of how best to ensure such a state in all species. Published research on environmental enrichment for nonhuman primates now fills volumes of journals and is catalogued periodically for ease of use by such groups as the Primate Information Center and the Animal Welfare Information Center. Despite this vast body of knowledge, questions still remain about the means and benefits of enriching the environment of primates as well as other animal groups. For example, social caging of the various species of nonhuman primates continues to present challenges (Reinhardt 1994; Ruppenthal and others 1991). Even in laboratory rats and mice, preferences for cagemates and environmental stimulation are not clear, and the ability of these provisions to enhance animal well-being is not proven (Chamove 1989; Chmiel and Noonan 1996; Spinelli 1990).

As our research has shown, environmental enrichment can have profound effects on behavior, on the anatomy of the nervous system and other organs, and on the immune system (as Capitanio describes in this issue in "Social Experience and Immune System Measures in Laboratory-housed Macaques: Implications for Management and Research"). This suggests that enriching the environment of laboratory animals would affect their well-being. As Sapolsky (1990) points out, however, some of the short-term measures of an animal's response to environmental changes may not have long-term consequences on animal well-being. Our studies show that rats given an opportunity to explore in an enriched environment gain weight more slowly than those in conventional caging; however, studies in nonhuman primates (Bayne and others 1991; Schapiro and Kesel 1993) and at least 1 study in mice (Chamove 1989) have shown faster growth rates. These effects as well as structural brain changes may be more enduring than the animal' s response to an immune system challenge. However, they require one to speculate on the relative influence on animal welfare of factors such as growth, body weight gain, and learning in a given species at a given developmental stage.

Behavioral Effects of a Nonstimulating Environment

In many species, environmental deprivation may lead to pathologic behaviors such as self-mutilation, fur/feather pulling, or coprophagy; and numerous studies have provided compelling evidence that enrichment can often reverse these problems (reviewed by Wemelsfelder 1990). Certain behaviors such as cross-suckling in farm species may not indicate a pathological condition but are detrimental to production goals. Morrow-Tesch (1997) has shown reduction of such behaviors by enriching postweaning enclosures. Dantzer (1986) discusses the central mechanisms of stereotypic behavior as a result of environmental deprivation in farm animals but makes no claims that animals exhibiting these behaviors are, in some way, suffering. Gone (1986) also finds well-being in farm animals difficult to assess but concurs with others that minimizing problem behaviors by improving a specific housing system is a worthwhile starting point.

Stereotypies or pathological behavior correlated with a lack of environmental stimulation are not often described in more common laboratory species; in fact, many consider certain small species exceptionally well-adapted to a laboratory environment. This is often cited in animal use protocols as a reason for choosing given laboratory species such as rodents or rabbits. Despite decades of research on environmental effects on the neuroendocrine system, at the time of this writing, comparative research indicating that environmental enrichment is essential to the general well-being of rodents has been inconclusive (see McGlone and others, "Floor Space Needs for Laboratory Mice: C57BL/6 Males in Solid-bottom Cages with Bedding," in this issue). Although Spinelli (1990) includes rodent studies when listing benefits to animals of environmental enrichment, he cites research published by our colleagues showing enhanced neuroanatomic and cognitive development as a result of enrichment. Certainly, as humans we often consider improved intelligence a reasonable measure of well-being, especially after more basic physical and emotional needs have been addressed. In wild animals, increased brain capacity is likely to confer some increased survivability. However, for a laboratory animal with no need to locate food, defend itself, or escape predators, the value of this effect on well-being is less obvious.

Wemelsfelder (1990) describes the result of environmental deprivation as boredom and proposes that animals that are exploratory by nature but cannot meet this behavioral need in a laboratory environment are likely to exhibit sensory deprivation behaviors. The Institute for Laboratory Animal Research (ILAR¹)publication Recognition and Assessment of Pain and Distress in Laboratory Animals (NRC 1992) specifically poses the question of whether lack of environmental enrichment clearly leads to distress in laboratory animals other than nonhuman primates. Although anecdotal reports indicate that returning an isolated rat to a group cage disrupts stereotypic somersaulting in rats, we found few published reports of controlled studies that reversed abnormal behavior in rodents by enriching the cage environment. Thus, although our research proves that drastic changes in environment, even for short periods, produce clear morphological changes in the nervous system, practical means of enriching animal caging for common research species may not elicit such changes and are not clearly a predictor of enhanced well-being.

Proposed Enrichment of Rodent Housing in a Typical Research Facility

Because the need for environmental enrichment in higher mammals has been accepted by scientists, facility directors, research funding agencies, and federal legislators, research in nonhuman primates has progressed to extensive preference testing to determine which means of environmental enrichment best accomplishes established goals such as minimizing pathological behavior and maximizing species-typical behavior (Bloomsmith and others 1991). Strategies for environmental preference testing have also been undertaken for farm (reviewed by Gonyou 1986) and zoo animals (Markowitz and Line 1990). However, as discussed by Gonyou (1986) and Fraser (1996), the limitations of preference tests may be particularly critical in evaluating methods for improving animal well-being.

Chamove (1989) investigated the effects of cage complexity on "emotionality" in mice by using such measures as home-cage activity; latency to emerge; ambulation, defecation, and grooming in an open field; adrenal weights; weight gain; and complexity preferences. Caging was made more complex by the addition of vertical walls producing alleyways, which the author compares with burrows. In these conditions, young mice gained weight more rapidly in the more complex cages despite increased home-cage activity levels. Although the tests appear to indicate a preference for the more complex cages, these were only 8-min tests. Controls for prior experience, novelty, or length of time needed to explore a much more complex cage were not described.

More recently, Chmiel and Noonan (1996) studied preferences of rats for intracage objects but found few significant preferences over a barren cage for the many objects (vertical walls, open-ended cans, pipes, wooden objects, and so forth) chosen to encourage species-typical behaviors (such as thigmotaxis, burrowing, gnawing) in rats. Anzaldo and others (1995) tested rats' preferences in cage configurations that might allow higher density caging in space missions and, contrary to Chmiel and Noonan, found a clear preference for the additional vertical walls anticipated for thigmotaxic animals. This supports our anecdotal observations that rats in enriched environments huddle in tight comers and niches when not actively exploring. Chmiel and Noonan did find a significant preference for some wooden blocks with holes and for small golf and wooden balls, but not for all wooden or apparently chewable objects. The adult male Long Evans rats used in this study were of the same sex and strain as most of those used in our research, and their age/weight range is compatible with those we have used to show plasticity in neural tissue of rats exposed to novel, enriched group caging. The authors suggest wood source hardness or palatability differences and the instability of some objects in these tippable preference cages as potentially negative factors. Although they also list neophobia as a possible factor, this would suggest that rats need more than a few days to develop a preference for an otherwise attractive object. As 1 author points out, this would present a significant challenge in developing or assessing any enrichment strategy for laboratory rats (Noonan 1997 personal communication). In general, however, Chmiel and Noonan's study supports inclusion of rats with macaques (Reinhardt 1997) and rabbits (Huls and others 1991) as a species that will spend significant time and may benefit from gnawing if provided appropriate objects.

Chmiel and Noonan's results differ from ours. Although most of our work has been conducted with weanlings, we have also found that adult rats, after 5 to 8 days of "socialization'' that typically includes substantial fighting, explore and interact with the environment and with each other in a manner less intense, but nonetheless very active, than during the postweaning "play bursts" of younger animals. Brain effects are smaller but otherwise quite similar to those seen in weanlings (for example, Green and others 1983; Juraska and others 1980). It may be that the exposure period in the Chmiel and Noonan study was simply not sufficient for this second phase of adaptation to the novel housing conditions to occur. It is important to note that previously isolation-housed rats are much more persistently aggressive in their adaptation to group housing in the complex environment.

Companionship may serve as a form of environmental enrichment, and its benefits may be quite evident. Group housing appears to allow for expression of species-typical behaviors and improved health in many species (Gonyou 1986; Grandin and others forthcoming; Huls and others 1991) including nonhuman primates, and certain species (such as dogs) almost certainly benefit from human companionship. The recently revised ILAR Guide for the Care and Use of Laboratory Animals (Guide1)(NRC 1996) includes strong recommendations to consider the social needs of various species in establishing housing procedures. Thus, understanding the effects of social contact on well-being is essential for evaluating these plans as called for in the Guide.

Although strict evaluation of synapse formation and other neuromorphological change is an impractical means of determining the value of an enrichment strategy for common laboratory species, we support attempts to improve animal well-being and acknowledge the implications of enhanced neural development associated with well-being. Given the extreme differences we see between our EC rats and those raised in the conventional yet relatively impoverished cages typical of modern research facilities, we would caution against acceptance of statements that scientific goals will not be affected by altering housing conditions (Chmiel and Noonan, 1996). In fact, as noted above, specific morphological, physiological, and behavioral differences induced by housing conditions can have direct potential impact on a variety of experimental procedures. Moreover, we and presumably many other research groups dependent on large numbers of caged mammals for our studies recognize the importance of using emotionally healthy and neurologically "normal" animals. At the risk of having to qualify comparisons with older studies of isolated, environmentally-deprived animals, future research should concentrate on using animals whose well-being is considered as critically important as their nutrition, cage sanitation, and health status.

¹Abbreviations used in this paper: AC, acrobat condition; EC, environmental complexity; FX, forced exercise; Guide, Guide for the Care and Use of Laborator3' Animals; IC, individual cage; ILAR, Institute for Laboratory Animal Research; LX, lack of exercise; SC, social condition; VX, voluntary exercise.

ACKNOWLEDGMENTS

Work toward preparation of this manuscript was supported by National Institutes of Health grants MH35321 and AG10154.

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