Which of the following is not a reason that animals are used in behavioral research?

Scientists use animals to learn more about health problems that affect both humans and animals, and to assure the safety of new medical treatments. Some of these problems involve processes that can only be studied in a living organism. Scientists study animals when there is no alternative and it is impractical or unethical to study humans.

Animals are good research subjects for a variety of reasons. They are biologically similar to humans and susceptible to many of the same health problems. Also, they have short life-cycles so they can easily be studied throughout their whole life-span or across several generations. In addition, scientists can control the environment around the animal (diet, temperature, lighting, etc.), which would be difficult to do with people. However, the most important reason why animals are used is that it would be wrong to deliberately expose human beings to health risks in order to observe the course of a disease.

Animals are needed in research to develop drugs and medical procedures to treat diseases. Scientists may discover such drugs and procedures using research methods that do not involve animals. If the new therapy seems promising, it is then tested in animals to see whether it seems to be safe and effective. If the results of the animal studies are favorable, human volunteers are asked to take part in a clinical trial. The animal studies are done first to give medical researchers a better idea of what benefits and complications they are likely to see in humans.

The behavior of living organisms is a visible manifestation of activity of the central nervous system. Thus, the study of behavior is a central feature of contemporary neuroscience research in animals. In some studies, the research emphasizes behavior itself, and the primary goal is to characterize behavior and its environmental determinants. In others, the behavior of an animal may be correlated with measurement of brain electric or chemical activity to understand brain mechanisms underlying behavior. Behavioral measures are also used often to detect or measure changes in brain function that may be produced by disease, neural injury, genetic modification, or exposure to various agents and treatments.

The purposes of this chapter are to address several general issues that arise in behavioral studies and to give more detailed consideration to a few specific aspects of neuroscience research in which the measurement of behavior is a central feature.

BEHAVIORAL STRESSORS

Some neuroscience research involves exposing animals to behavioral stressors. These manipulations can be social (such as involving social separation or mixing of unfamiliar animals) or nonsocial (such as exposing animals to novel environments or restricting behavioral activity).

This research focuses on three avenues of investigation. The first is aimed at understanding the effects of exposure to behavioral stressors on aspects of neural function or conversely understanding how neural manipulation affects responses to behavioral stressors (von Borrell, 1995). For example, a pregnant monkey might be exposed to various behavioral stressors, such as noise and unfamiliar surroundings, and neurochemicals associated with the stress response would be measured in her offspring to determine the effects of prenatal stress on the development of stress responsiveness in young animals (Schneider et al., 1998).

The second is aimed at understanding the neural substrates or correlates of particular behaviors or aspects of temperament, including social recognition, affiliation, pair bonding and attachment, parental behavior, social dominance, aggression, predation, play, and fearfulness (Amaral, 2002; Kavaliers and Choleris, 2001; Siegel et al., 1999; Young, 2002). In those studies, animals may have lesions, be genetically modified (mouse knockouts), or be electrically or chemically stimulated, and the resulting behaviors can be observed; or neural function may be measured during or after the performance of the behaviors of interest.

The third category consists of pharmacologic studies to determine the efficacy of various compounds in reducing aggression, anxiety, or fearfulness (Mench and Shea-Moore, 1995). The purpose of those studies is usually to identify compounds that may be useful in human or veterinary clinical medicine, but pharmacologic testing can also be used for studies of underlying mechanisms of behavior: the behavior of interest is stimulated in some way, usually by staging an aggressive encounter or placing an animal in a fear-inducing situation, and compound efficacy is then evaluated with behavioral measures.

Social Disruption

Social disruption can be used as an experimental technique in neuroscience and behavioral research, but it can also be an inadvertent confounder of the research. Experimental designs that purposefully incorporate social disruption, do so through the temporary removal and reintroduction of offspring or of group or pair-mates, longer-term or repeated reorganization of social groups by removal of group members or by introduction of unfamiliar animals to groups or to one another, or even the merging of different groups of animals. Abnormal social conditions can also be created by placing animals in atypically small or large social groups, by forming groups of atypical composition (such as all-male groups or groups comprising only animals of similar age), or by crowding them. In addition to the study goals described above, this technique has recently been used to study coronary artery atherosclerosis, heart rate reactivity, and the effects of exercise in conjunction with social disruption on coronary heart disease (Kaplan et al., 1982, 1993; Manuck et al., 1983a,b; Williams et al., 1991, 2003).

The effects of social separation (such as individual housing) or social isolation on an animal's behavioral profile have been documented in various species. The impact of social separation or isolation can depend on the species or strain of animal, the age at which an animal is removed from conspecifics, the duration of the separation, and the completeness of the separation (with respect to visual, auditory, or olfactory cues from other animals). In nonhuman primates, the lack of physical contact appears to be the most important cause of abnormal behavior, both in infants and in adult animals (Bayne and Novak, 1998).

Animals that are isolated to disrupt the infant-parent bond often display acute responses to indicate stress. Distress vocalization, changes in general activity and heart rate, as well as elevated cortisol/corticosterone concentrations can occur and are adaptive under normal circumstances. However, if the separation is prolonged, as during experiments where the effects of infant-parent bond disruption are being studied, it becomes distressful and can lead to maladaptive behaviors as the infant animal matures. Self-injurious behaviors, stereotypic behaviors, extreme timidity or aggressiveness, and inability to mate or provide adequate care to offspring are maladaptive behaviors that might result from the social disruption (NRC, 1992).

Kittens separated from their mothers at an early age tend to be more aggressive and nervous as adults (Seitz, 1959), and social play is critical for a kitten's development (O'Farrell and Neville, 1994). Puppies that are not adequately socialized to other dogs or people may be excessively fearful or aggressive (O'Farrell, 1996). Wolfle (1990) has described a puppy-socialization program and behavioral scoring method specifically for use in the research environment. Monkeys reared in partial or total social isolation develop a syndrome of behavioral abnormalities that includes rocking, huddling, self-clasping, and excessive self-orality (Cross and Harlow, 1965; Harlow and Harlow, 1965). As the animals age, stereotypic patterns emerge, such as repetitive locomotor patterns, floating limbs, and eye poke or salute. The isolation syndrome is also manifested in the development of abnormal social relationships (Mason, 1968).

A restricted social environment can also affect adult animals. For example, long-term (2-year) individual housing of adult nonhuman primates has been shown to alter social behavior (Taylor et al., 1998). Unless the research focuses on social restriction or veterinary concerns develop, infant animals should be reared in a social environment with mother and peers, with mother only, or with peers only to reduce or prevent psychopathologic conditions (Bayne and Novak, 1998). Similarly, when the research, health, and safety of the animals allow it, adult social animals should be maintained in a social environment (for example, pair- or group-housed).

Animal Care and Use Concerns

The primary animal care and use concern associated with social disruption is the distress that leads to the display of maladaptive behaviors. When studies involve the use of social disruption, the animal-use protocol should include humane endpoints for removal of the animal from the study. Determining endpoints that are predictive of severe distress is a matter of professional judgment and should evolve through discussions between the IACUC, veterinarian, and PI.

It is important to recognize that the display of the maladaptive behavior affects not only the isolated animal but can also have unintended affects on the dam (in studies of infant-parent bonds), the potential offspring of the animal, and the conspecifics that may be forced into the animal's social group. In some species, such as nonhuman primates, dams also show a response to separation from their infants. Their behavioral and physiologic reactions appear to be similar to those of the infant, although less persistent and intense (NRC, 1992), and steps should be taken to minimize this distress if it is an unintended byproduct of the experiment.

The offspring of animals in social disruption experiments may also be impacted by the maladaptive behavior of its dam. For example, female rhesus macaques that are isolate-reared can be neglectful or abusive of their infants (Suomi, 1978). In that situation, it may be appropriate to provide additional support to the offspring or protect it from injury.

In some cases, social disruption causes aggression toward conspecifics. For example, social restriction of male mice will lead to intermale fighting (Brain, 1975). Similar findings have been observed in gerbils, hamsters, and rats (Karim and Arslan, 2000; Payne, 1973; Wechkin and Breuer, 1974). Isolation-reared rhesus monkeys are hyperaggressive and do not develop normal social relationships with other monkeys (Anderson and Mason, 1974; Mason, 1961); this aggression can be directed to other animals or be self-directed (Gluck et al., 1973). Steps should be taken to prevent injury in these cases. For instance in nonhuman primates, this may require housing the aggressive animal separately (AWR 3.81(a)(1)) or the use of screen barriers within cages to permit side-by-side contact, but prevent agonistic encounters.

Induced Aggression or Predation

Several common models are used in studies whose primary intent is to induce aggression or predatory behavior (Mench and Shea-Moore, 1995):

• Isolation-induced aggression. This involves isolating a male mouse or rat for several weeks and then staging a brief encounter (usually 5–10 minutes) with an unfamiliar group-housed male. Encounters may be staged either in the isolate's cage or in a neutral arena. If drugs are administered, they may be administered either to the isolate or to both animals. Because cues from the introduced animal can affect the outcome of the encounter, introduced mice are sometimes rendered anosmic before testing to make them less responsive to social stimulation (Stowers et al., 2002).

• Naturalistic paradigms. These studies aggression by placing animals in circumstances that approximate the situations that they might encounter in the wild, where they have to compete for resources, defend territories, or integrate into new social groups. Examples are introducing an ‘intruder” animal into the cage or enclosure of a group of resident animals (Blanchard et al., 1975), mixing two social groups by removing a partition between their cages (Zwirner et al., 1975), and requiring animals to compete for access to food by displacing one another from a tunnel (Miczek, 1974). Isolation of mice is not necessary to study aggression; pair-housing of a male with a female promotes consistent aggressive behavior when the male is tested in a resident-intruder situation (Fish et al., 2002). Animals may also simply be observed in their normal social groups, either in the laboratory or in the wild; this process is facilitated by the use of osmotic minipumps to deliver neuromodulators or hormones and radiotransmitters for remote collection of physiologic data.

• Aggression modified by drugs. Using an “intruder” paradigm, it has been shown that drugs, such as alcohol and allopregnanolone (a positive modulator of the GABAA receptor) can increase the expression of aggressive behavior in mice (Fish et al., 2002). In contrast, other drugs, such as 5-HT1B agonists (for example, anpirtoline) will inhibit the expression of aggression (de Almeida and Miczek, 2002).

• Predatory aggression. This involves introducing prey species to animals, especially introducing rodents to cats and mice to rats (the muricide model). If the object of the research is to understand or influence the full predatory sequence or if the sequence ensues so rapidly after initial attack that intervention is not possible, death of the prey animal is often the endpoint. Because pain and injury to both the prey animal and predator are significant welfare issues with these kinds of studies, methods to protect the prey animal from physical attack or modeling elements of the predation sequence should be considered (Novak et al., 1998b). It may not even be necessary to use live prey. The number of times an animal serves as prey should be limited. The use of wild caught animals may be preferred due to their potentially greater experience and skill in predatory avoidance (Novak et al., 1998b). In those instances where the prey animal dies, the study should be designed to expedite the predation sequence and to minimize the pain and distress experienced by the prey animal (Huntingford, 1992).

Any situation in which unfamiliar animals are mixed or established social groups are perturbed has the potential to result in aggression, whether or not aggression is central to the aims of a study. The effects of the aggression on the recipient animal will depend on the intensity, duration, and potential for injury associated with the aggression and hence on the species being studied, the ages and sexes of the animals, and their past social experiences. If aggression is incidental to the goal of the study, many methods can be used to reduce the potential for injury, including gradual introduction of animals, allowing partial contact (for example, visual, auditory, olfactory, or tactile) before mixing and providing refuge areas to which introduced animals can escape from aggressors (Bayne and Novak, 1998). Naturalistic approaches to inducing aggression or predation may not only minimize injury but also provide information that is more reflective of the range and types of behaviors shown by animals under more ecologically relevant circumstances (Kavaliers and Choleris, 2001; Mench and Shea-Moore, 1995).

Even when aggression is a desired outcome of a study, attention should be given to minimizing injury and distress (Anonymous, 2002; Bayne and Novak, 1998; Ellwood, 1991; Huntingford, 1984). Ways of doing that include minimizing the numbers of animals used; decreasing the length of an encounter to the shortest time necessary to collect the required information, which may involve continuous observation with intervention to stop aggression at predetermined points; using artificial “model” animals rather than real animals as the recipients of aggression or the initiators of predatory encounters; placing introduced animals behind protective screens (for example, Habib et al., 2000) or barriers (Perrigo et al., 1989); and allowing the introduced or subordinate animal to control the intensity of aggression by providing refuge areas. Each of those strategies has limitations, and their usefulness will depend on the species being studied and the purpose of the study. Animals that are severely injured during an encounter should be removed as soon as possible and treated or euthanized. The use of specific animals as targets of prolonged aggression should be well justified.

Environmental Deprivation

Animals may be exposed to nonsocial behavioral stressors to determine their effects on neural and neuroendocrine function. For example, animals may be restrained for brief or for sustained periods by being held, tethered, chaired, or immobilized by other restraint devices or placed in small enclosures or wrappings that restrict movement. Restraint may be repeated at intervals to cause intermittent stress. The animal-welfare issues associated with restraint are discussed in Chapter 3 (“Physical Restraint”).

In other studies, the behavior of animals is restricted by placing them in barren environments that provide few opportunities for normal behaviors or by restricting sensory input. One or more sensory modalities (touch, audition, vision, and olfaction) may be restricted, or animals may even be kept in complete sensory isolation. The goal of such studies is generally to determine the effects of restricted environmental input on neural development. Restricted sensory or behavioral input often leads to the development of severely abnormal behaviors. Whether these effects are reversible depends on the species, the duration of restriction, and the age at which the animals are restricted. Consideration should be given to the impact of this type of research using long-lived animals due to the protracted and resilient behavior changes invoked.

Environmental Stimulation

Stress can be induced by exposing animals to novel or extremely complex environments. The emphasis is usually on neural development, generally with a focus on fear and exploratory behaviors. Fear and exploration may be assessed with a standard battery of tests, some of which are described earlier in this chapter. Extreme novelty or complexity can have adverse physiologic and behavioral effects. However, moderate novelty and species-appropriate complexity actually have generally beneficial effects, such as enhancing neural development, learning and spatial ability, and stress competence. This is reflected in the AWRs (3.81), which mandates an appropriate plan for environmental enhancement adequate to promote the psychological well-being of nonhuman primates.

The purposeful use of environmental stimulation for experimental reasons should be distinguished from incidental, but no less stressful, stimuli that may occur in an animal facility and impact ongoing research. In either case, young animals are more susceptible to a prolonged effect of environmental stimulation and thus the use of long-lived species in this research should be well justified if the intention is to maintain the animals in the colony for extended periods of time.

Animal Care and Use Concerns

The goal of many studies involving behavioral stressors is the induction of stress responses. Exposure to intense, repeated, or prolonged stressors can have a variety of adverse effects, including suppression of reproduction, immune dysfunction, cardiovascular and gastrointestinal impairment, and persistent disruption of neuroendocrine function (Moberg and Mench, 2000). One consequence of exposure to behavioral stressors may be the development of abnormal behaviors, including self-mutilation, mutilation of other animals (such as tail-biting in pigs and cannibalism), and stereotypic behaviors (such as bar-chewing or route-tracing). Causative factors of abnormal behaviors include social isolation, rearing in a barren environment or lack of sensory stimulation, and excessive environmental or social stimulation. Once developed, the behaviors tend to persist even when the original eliciting stimulus is removed, so the animals in question may have special husbandry and care requirements. Minimizing the duration, frequency, and intensity of stressors can minimize the effects.

When young animals are separated from their dams, parents, or broader social groups for experimental purposes, provisions must be made to care for the animals, both physically and behaviorally. In some cases, partial socialization (either temporally or physically limited contact) with peers or compatible species may be possible to mitigate the immediate stress imposed by the socially restricted environment and to improve the long-term behavioral health of the experimental animals. Alternatively, separation may be delayed until the animals are older to limit the effect of restriction. Novak et al. (1998a) suggest that young animals be monitored closely and evaluated regularly if they are separated, thus enabling more informed management decisions to address the animals' well-being.

Animal-use protocols for research involving behavioral stressors should include a thorough description of the potential animal-welfare issues associated with each stressor and a detailed plan for monitoring, record keeping, and determining when to end a test early to avoid unnecessary pain and/or distress. If little or nothing is known about the possible outcomes of exposure to a particular behavioral stressor, IACUC review and approval of the protocol may involve a requirement to conduct pilot studies, mandatory oversight of initial testing by veterinary staff, or provision of regular progress reports as a condition of continuing approval.

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