National Academies Press: OpenBook
« Previous: 8 Agents and Treatments
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

9
Behavioral Studies

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.

USE OF APPETITIVE AND AVERSIVE STIMULI

Terminology

Stimuli that can be labeled appetitive (attractive or pleasant) or aversive (noxious or unpleasant) are often used in behavioral research. Such stimuli may include food pellets, sweet or bitter tasting solutions, loud noises, drugs, or electric shock. Because use of such stimuli, especially aversive stimuli, is sometimes a source of concern in behavioral studies, this section begins with a brief discussion of the ways in which behavioral neuroscientists commonly describe

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

and categorize these events. In general, appetitive stimuli are ones that an organism will voluntarily make contact with or approach, and aversive stimuli are ones that an organism will try to escape or avoid. Central to those definitions is the idea that the labeling of a stimulus as appetitive or aversive is based on an organism’s behavior, not on physical features of the stimuli themselves. Indeed, the same stimulus may be appetitive in some situations or to some individuals, but aversive in other situations or to other individuals. For example, in certain behavioral procedures, rats and monkeys have been shown to engage repeatedly in behaviors that produce exposure to electric shock, an event commonly assumed to be an aversive (Brown and Cunningham, 1981; Cunningham and Niehus, 1997; Cunningham et al., 1993; Kelleher and Morse, 1968). Thus, under these experimental conditions, electric shock would be labeled an appetitive stimulus, not an aversive one. Similarly counterintuitive examples can be found in the literature on behavioral effects of abused drugs. For example, the same dose of alcohol that produces a conditioned place aversion in rats will produce a conditioned place preference in mice (Cunningham et al., 1993). Moreover, a drug’s ability to produce a conditioned preference may be completely reversed (to conditioned aversion) simply by changing the temporal relationship between drug injection and the associated stimulus (e.g., Cunningham et al., 1997; Fudala and Iwamoto, 1990). It has also been shown that injection of an abused drug may concurrently induce preference for a paired spatial location, but aversion for a paired flavor solution in the same animal (e.g., Reicher and Holman, 1977). All of these examples illustrate that decisions about whether a given stimulus should be considered appetitive or aversive cannot be based solely on its physical properties, but must be informed by expert knowledge of its behavioral effects in various contexts. Importantly, those effects may vary significantly as a function of the species, genotype, sex, and past experience of each animal.

In more technical terms, the stimuli under consideration here are often referred to as either reinforcers or punishers, depending on their effects in behavioral procedures in which the response-contingent presentation or removal of a stimulus produces either increase or decrease in the rate of the target response. Stimuli that increase the rate of a contingent behavior are called reinforcers, whereas events that decrease the rate of a contingent behavior are called punishers. Both reinforcement and punishment may involve either the presentation or the removal of a stimulus (Domjan, 1998). Typically, the response-contingent presentation of an appetitive stimulus produces an increase in responding (positive reinforcement) and the response-contingent presentation of an aversive stimulus produces a decrease in responding (positive punishment). In contrast, the response-contingent removal or omission of an aversive stimulus produces an increase in responding (reinforcement based on escape or avoidance), whereas the response-contingent removal or omission of an appetitive stimulus produces a decrease in responding (punishment based on omission training).

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

Although the foregoing definitions indicate that evaluation of behavioral changes produced by response-contingent delivery or removal of such events is critical for applying the labels, the manner in which the stimuli are used in experiments may or may not involve a response contingency. For example, in studies that use instrumental learning or operant conditioning, there will be an explicit, experimenter-defined relationship between some feature of the animal’s behavior (such as whether a lever is pressed) and the delivery (or withholding) of the stimulus. In contrast, studies that use classical or Pavlovian conditioning typically do not involve a response-outcome contingency; rather, the emphasis is typically on the predictive relationship between some other stimulus (such as a light or tone) and delivery of the appetitive or aversive stimulus (Rescorla, 1988). Because brain mechanisms underlying the different types of learning may differ, the decision to present appetitive and aversive events in a response-contingent or response-noncontingent manner should be based on the scientific goals of the study.

Rationales for Using Appetitive and Aversive Stimuli

Appetitive or aversive stimuli are typically used to motivate an animal to perform a particular behavior. However, the scientific reasons for producing that behavior can vary widely, and the overall purpose of the study will be an important consideration in the selection of the appetitive or aversive stimuli. For example, a considerable body of neuroscience research using appetitive and aversive stimuli focuses on understanding the neurobiology of basic motivational processes, such as those involved in feeding, drinking, foraging, mating, drug addiction, aggression, fear (anxiety and phobias), and the avoidance of pain or discomfort. In such cases, the selection of a particular motivational stimulus (such as salt water or a sexually receptive conspecific) is typically dictated by the specific motivational or behavioral system under study (such as sodium appetite or copulation). In other cases, however, investigators may have more leeway in the selection of motivational stimuli. For example, investigators interested in the general neural mechanisms underlying simple learning (such as classical and operant conditioning), cognition, or memory may be able to use a range of stimuli, both appetitive and aversive, to achieve their scientific aims. Similarly, investigators who simply wish to establish a reliable behavioral baseline for studying motor, sensory, attentional, or perceptual processes or for assessing the effect of various manipulations may also have some flexibility in their choice of motivational stimuli. Relevant considerations might include whether the stimulus has similar effects among species or among individuals within a species. Another consideration is the degree of variability in the efficacy of the stimulus among individuals or of repeated exposures to the stimulus in the same individual. For example, because of rapid satiation, a food rich in calories will be a poor choice as a reinforcer in a procedure that requires the animal to respond repeatedly for

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

food over a period of several hours. Of course, the choice of motivational stimuli in such experiments will also be guided by appropriate consideration of their potential to cause pain or distress. This issue is addressed in more detail in the next section.

Animal Care and Use Concerns

As in other types of research with laboratory animals, investigators conducting behavioral research must consider the recommendations in the Guide when making decisions about the choice of appetitive and aversive stimuli. In particular, consideration must be given to Principle IV of the US Government Principles (IRAC, 1985):

Proper use of animals, including the avoidance or minimization of discomfort, distress and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals.

Neuroscientists proposing to use appetitive or aversive stimuli should provide a clear and complete description of the characteristics of the stimulation (such as unit amount, concentration or intensity, duration, and total number) and a scientific rationale for their use in their animal-use protocols. Due consideration must be given to the immediate consequences of acute exposure to these stimuli (for example, do they cause more than momentary or slight pain or distress?) and to possible detrimental effects of long-term or repeated exposure (for example, development of dental caries after prolonged exposure to sugared foods or fluids). Consideration must also be given to possible adverse consequences of restricted access to food or fluids that may be required to provide an appropriate motivational state for the appetitive stimulus (see Chapter 3). As noted earlier, selection of the specific motivational stimuli in a task may be influenced by limitations imposed by the recording techniques; for example, an event that produces little or no movement in an animal may be preferred in sensitive physiologic recording procedures. In some situations, choice of a motivational stimulus and its characteristics will be guided by previous research showing that variability in response to it is low, thus reducing the number of animals that must be used in the procedure. It is also possible that the characteristics of the event must be adjusted individually for each animal to maximize its efficacy or minimize its detrimental effects.

General strategies used by the IACUC, veterinary staff, and members of the research team to evaluate the choice of appetitive and aversive stimuli should mirror those described in previous chapters. In the case of aversive/punishing stimuli with the potential to cause pain and distress, the evaluation process described in Chapter 2 (“Pain and Distress”) can be used. As noted earlier, generally

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

acceptable levels of noxious stimulation are those that are well tolerated and do not result in maladaptive behaviors. Use of aversive stimuli at intensities or durations that approach or exceed the animal’s pain tolerance level should generally be avoided in behavioral procedures, unless a scientific justification is provided. As discussed previously, it is important to note that the appearance of escape and avoidance behaviors may occur well before the intensity of a stimulus reaches the pain tolerance level. In such cases, these behaviors would be considered appropriate adaptive responses. It is only when the animal’s behavior is dominated by escape-avoidance attempts that the behavior becomes maladaptive, signaling unacceptable levels of pain (NRC, 1992).

At first glance, one might assume that avoidance or minimization of discomfort, distress, and pain is more problematic when aversive stimuli are used to motivate behavior than when appetitive stimuli are used. However, that is not necessarily true, especially when one considers that the efficacy of some appetitive foods and fluids depends on the introduction of a restricted schedule of access to food or water (see Chapter 3). Thus, in some situations, an aversive stimulus that does not require prior induction of a “need” state (such as contact with mild electric shock or placement in a pool of water) may actually produce less overall discomfort and distress than the combination of an appetitive stimulus with food or fluid restriction. At the same time, however, one must recognize that detection and measurement of “distress” in animals remains problematic (NRC, 2000) (see “Pain and Distress” in Chapter 2).

In some cases, an investigator’s choice of a particular appetitive or aversive stimulus will be determined by scientific reasons. For example, the choice of aversive stimulation such as exposure to electric shock or a predator could be justified by a specific scientific interest in understanding the brain mechanisms underlying behaviors motivated by fear or anxiety. In other situations, however, the scientific question may not directly dictate the choice of one type of stimulus over another. For example, the scientific goals of investigators interested in the neural bases of learning and memory or the mechanisms underlying a specific type of motor behavior might be accomplished by using a broad range of appetitive or aversive events. In situations where the scientific rationale for the choice of a particular motivational stimulus is not compelling or the IACUC is unsure whether one stimulus produces more or less overall discomfort or distress than another (e.g., mild electric shock versus a food pellet combined with food restriction), a useful strategy may be to allow the research to begin using the investigator’s preferred stimulus, but to agree in advance to a joint plan for rigorous monitoring and periodic re-evaluation by the IACUC. If apparent pain or distress is higher than expected or other adverse consequences are noted, stimulus parameters can be refined or the stimulus choice changed with approval by the IACUC. If no problems arise during the monitoring phase, the protocol may continue as originally proposed.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

An example of the issues that must be considered when evaluating the selection of a behavioral task can be provided by comparing the features of three different procedures commonly used to study spatial learning and memory in rodents: the radial arm maze (Olton and Samuelson, 1976), the Morris water maze (Morris, 1981), and the Barnes circular platform maze (Barnes, 1979). A growing interest in understanding the cognitive decline that accompanies aging together with the recent increase in the number of mouse models carrying genetic mutations thought to affect brain function has encouraged many investigators to use one or more of these tasks to assess “cognitive” function. Although many of the behavioral and brain mechanisms involved in solving these tasks are thought to overlap, the motivational basis for performance differs significantly in each task. For example, the radial arm maze procedure usually involves food or water restriction to motivate animals to seek reinforcers placed at the end of each maze arm, requiring the IACUC to consider the issues discussed previously in Chapter 3 (“Food and Fluid Regulation”).

In contrast, the Morris water maze involves immersion in water at or a few degrees above room temperature to motivate animals to swim to a hidden or visible platform. Because exposure to water has the potential for evoking a stress response, the time an animal is in the water should be minimized. Moreover, consideration must be given to drying the animal and providing access to an appropriate heat source (unregulated heating pads and heat lamps should be avoided as they can develop hot spots and cause thermal burns) after water exposure to prevent hypothermia. In the Barnes circular platform maze, the animal is typically placed on a large open platform in a well-illuminated room. The behavior of finding the hole that leads to the darkened escape tunnel located beneath the platform is presumably motivated by the animal’s natural aversion to bright open spaces. Some investigators have suggested that this task produces less stress in rats than tasks involving water immersion or food restriction (e.g., McLay et al., 1998). However, the procedures used in several recent studies suggest that additional aversive stimulation (e.g., intense lights, loud sounds, air stream from an overhead fan) may be required to adequately motivate mice to perform in the Barnes maze (Pompl et al., 1999; Inman-Wood et al., 2000; King and Arendash, 2002; Zhang et al., 2002). Thus, at least in mice, this task has the potential to evoke a stress response that may be similar to or greater than that evoked by the other two tasks.

In the case of lesioned or genetically modified animals, the choice of task may be further complicated by sensory-motor impairments that could increase the likelihood of stress or serious injury (e.g., drowning in the water maze, falling off the edge of a Barnes maze). As suggested above, when there is uncertainty about which task produces the least amount of discomfort or distress while still meeting the investigator’s scientific goals, the IACUC’s best strategy may be to work with the investigator to develop a thorough plan for monitoring the impact

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

of the procedure in conjunction with frequent re-evaluations by the IACUC until the consequences of the procedure are better understood and shown to be acceptable.

BEHAVIORAL SCREENING TESTS

Behavioral screening tests are used in pharmacology, genetics, and health surveillance (health surveillance through evaluation of animal behavior is discussed in “Using Animal Behavior to Monitor Animal Health” in Chapter 2). Behavioral screening tests differ from hypothesis-driven experiments in that screening tests assess multiple behavioral measures because there is little information to indicate what important effects might be observed. Screening also is used if limitation of time and resources require a test that can be administered quickly to a number of animals. Screening tests are usually directed at broad functional domains, such as motor coordination, emotion, or sensory functions.

Neurobehavioral screens were developed more than 25 years ago to study pharmaceutical and chemical agents (Kulig et al., 1996; Moser, 2000b; Ross et al., 1998). Similar methods are used to screen for genetic mutants (Crabbe et al., 1999b; Sarter et al., 1992a,b; Warburton, 2002).

In reviewing the history of behavioral screening, Warburton (2002) considers the advantages of quantitative methods versus the simplicity of observational screening methods. Observational methods are especially appealing to those with little experience in behavioral science, who may not focus on the possible limitations of observational methods: subjective interpretation, higher variability of baseline behavior, and observational variation among observers. Screening results are most useful if one can demonstrate between-observer reliability, establish standardized protocols, and validate the screen with “gold-standard” procedures.

Behavioral Screening in Pharmacology and Toxicology

Unlike research protocols for pharmacology and toxicology, drug screening usually implies that the effects of the test compound are not well known. Screening studies can be justified by the need to detect a chemical’s ability to cause health problems in humans or animals (such as the abuse liability of a new pharmaceutical or the neurotoxicity of an industrial product) or to determine whether its effects warrant more detailed investigations of its potential as a treatment for behavioral or neurologic disorders. Screening tests also are used when a drug’s pharmacokinetics are not well known and observations are required over an unknown time to determine whether an organism’s response to the drug changes during chronic exposure and whether such exposure can lead to physical dependence. The IACUC must be aware that regulatory agencies (such as the Environmental Protection Agency or the Food and Drug Administration) some-

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

times require investigators to use specific test methods and experimental designs (Weisenburger, 2001).

Behavior has proved to be a convenient experimental variable in screening because it is noninvasive and because alterations in many physiologic systems can be reflected in changes in behavior. The functional observational battery (FOB; see Table 9-1) (Moser, 2000a) is a systematic neurologic examination for rodents involving a neurologic examination with numerous behavioral measures. It provides more extensive behavioral measures than the mouse ethogram discussed in Chapter 3 (“Genetically Modified Animals”). The FOB procedure has also been adapted for use with weanling rats (see Table 9-2) (Bushnell et al., 2002; Moser, 2000a). Scoring of the FOB is semiquantitative, and the FOB should be administered and scored by an experienced technician. When a skilled technician is not available, or when handling the animals might be dangerous to animals or staff, observation of an animal in its home cage can be useful, particularly if a quantitative rating scale is used to document the appearance of abnormal behaviors. Better quantification is obtained with commercially available equipment, such as photocell arrays, than through direct observation. The equipment is placed outside a rodent’s home cage to measure activity, such as locomotion and rearing, and this avoids the necessity of handling the animal and the possibility that handling may cause changes in the animal’s behavior (Evans et al., 1986; Lessenich et al., 2001). Additional methods used in screening for neurotoxicity are reviewed by Weisenburger (2001).

Screening methods for nonhuman primates can be considered along a scale of intrusiveness into the nonhuman primate’s living space. Nonintrusive procedures are used to minimize handling the animals. Behavioral activity level, diurnal rhythms, etc., can be monitored with photocell arrays surrounding the home cage (Evans et al., 1989). A food-pickup test can also be used while a primate remains in its home cage. Small pieces of food (such as raisins and peanuts) are systematically placed on a tray and then moved to within the nonhuman primate’s reach. The observer measures the time taken to extract the food and the accuracy in terms of the number of attempts required to retrieve all of it (Evans et al., 1989; Merigan et al., 1982). That provides evidence of visuomotor coordination and appetite. Finally, if the experiment permits removing the nonhuman primate from its home cage to a special test apparatus (see “Restraint” in Chapter 3), video cameras can be used to remotely monitor nonhuman primates while they are in the special test apparatus (Ro et al., 1998). A battery of operant conditioning techniques have also been employed to assess neurologic changes caused by a drug or chemical in nonhuman primates (Schulze et al., 1988). This operant screening test is called the Operant Test Battery (OTB; see Table 9-3), and was developed at the National Center for Toxicological Research (NCTR). Additional studies have shown that the OTB can also be used to assess neurological effects in humans and rats (Paule, 2000, 2001).

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

TABLE 9-1 Functional Observational Battery for Adult Rats

Endpoint

Measurement/Scale

Pupil Response

present/absent

Abnormal Body Posture

present/absent

Piloerection

present/absent

Forelimb and Hindlimb Grip Strength

kg of force

Landing Foot Splay

cm/mm

Body Weight

g

Body Temperature

°C

Open-Field Rearing

number

Gait Score (also description of gait)

1 to 5

Ataxia Score

1 to 5

Aerial Righting Response

1 to 4

Home-Cage Activity

1 to 5

Open-Field Activity

1 to 6

Arousal

1 to 5

Ease of Removal

1 to 5

Handling Reactivity

1 to 4

Tremorigenic Score

1 to 4

Salivation

1 to 3

Lacrimation

1 to 3

Urination/Defecation

1 to 5

Tail-pinch Response

1 to 5

Click Response

1 to 5

Touch Response

1 to 5

Approach Response

1 to 5

 

SOURCE: Moser, 2000b.

TABLE 9-2 Functional Observational Battery for Pre- and Post-weanling Rats

Endpoint

Measurement/Scale

Body Weight

g

Open-Field Rearing

number

Gait Score (and description of gait)

1 to 3

Forelimb Grabbing

1 to 4

Surface Righting Response

1 to 4

Open-Field Activity

1 to 6

Arousal

1 to 5

Handling Reactivity

1 to 4

Tremorigenic Score

1 to 3

Urination/Defecation

1 to 5

Lacrimation

1 to 3

Salivation

1 to 3

Tail-Pinch Response

1 to 5

Click Response

1 to 5

 

SOURCE: Moser, 2000b.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

TABLE 9-3 NCTR Operant Test Battery

Function

Name of Test

Motivation

Progressive Ratio Task

Discrimination

Color and Position Discrimination Task

Timing

Temporal Response Differentiation Task

Short-term Memory

Delayed Matching-to-Sample Task

Learning

Repeated Acquisition Task

 

SOURCE: Paule, 2000.

An important consideration for the IACUC, researcher, and veterinarian is the selection of the animal species to be used for behavioral screening in pharmacology and toxicology (Luft and Bode, 2002). Available data on kinetics and metabolism should be taken into consideration in identifying a species whose behavior will best predict effects in humans. Generally speaking, rodents are good models for behavioral screening in studies of neurotoxicity and neuropharmacology (Luft and Bode, 2002).

Some behavioral-toxicology experiments involve dosing that produces deleterious effects. The protocol should provide a contingency plan for conditions in which side effects will be alleviated or that require an animal’s removal from an experiment (see “Animal Care and Use Concerns Associated with Toxicity or Long-lasting Drug Effects” in Chapter 8).

Behavioral Screening of Genetically Modified Animals
General Considerations

Once a general health assessment of a newly developed strain of genetically modified animals is completed (see “Genetically Modified Animals” in Chapter 3), behavioral phenotyping should proceed as soon as sufficient numbers of transgenic animals are available to identify sensory, motor, or motivational deficits that may compromise animal well-being. Sensory and motor assessments should be completed before assessment of more complex behaviors—such as learning and memory, aggression, mating, and parental behaviors—because sensory and motor deficits may confound the interpretation of other behavioral assessments.

Behavioral tests assess the effects of altering, adding, or removing a gene (and gene product) on behavior, not the effects of the normal gene on behavior (Nelson, 1997). Behavioral phenotyping can also be confounded by impairments that are secondary to the missing or inserted gene; for example, knocking out a gene may cause the compensatory overexpression of a second gene and any changes in behavior could be the result of the overexpression of the second gene. Those possible problems can be overcome in the same way as in other types of

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

ablation studies: by collecting converging evidence with a variety of pharmacologic, lesion, and genetic manipulations. Because mammalian genome mapping currently focuses on mice (Mus musculus), standardized behavioral testing of mice should be adopted (Brown et al., 2000; Crawley, 2000).

Altered behaviors of knockout mice are often sufficiently obvious or unusual that they catch the attention of animal-care personnel, who then notify the investigators. Dramatic behaviors that include increased aggression, altered maternal care, decreased sexual behaviors, seizures, and impaired motor coordination and sensory abilities are commonly reported for knockout mice (e.g., Barlow et al., 1996; Brown et al., 1996; Brown et al., 2000; Chen et al., 1994; Crawley, 2000; Nelson et al., 1995; Saudou et al., 1994). Presumably, knockout mice may have more subtle behavioral changes that have not yet been discovered, even among mutants with no obvious behavioral phenotypes. Some of the behaviors probably will be revealed only if the animals are housed in conditions that are ecologically relevant with respect to space and social organization (Cabib et al., 2000; Pfaff, 2001; Potts et al., 1991).

Behavioral performance is compared among wild-type (+/+), heterozygous (+/–), and homozygous (–/–) mice in which the gene product is produced normally, produced at reduced levels, or missing, respectively. The comparison of +/+ and –/– littermates of an F2 recombinant generation is probably the minimal acceptable control in determining the behavioral effects of knocking out a gene or genes (Morris et al., 1996).

In the past, many knockout strains were generated by using stem cells from one strain and blastocysts from another strain (see “Knockout and Knockin Mutants” in Chapter 3 for review). Therefore, behavioral differences shown by knockout mice may reflect strain effects rather than the effects of the absence of the missing gene (Broadbent et al., 2002; Gerlai, 1996; Threadgill et al., 1995). Given the potentially important effect of background genotype on ability to detect effects of targeted mutations (Crabbe et al., 1999a; Lariviere et al., 2001), behavioral neuroscientists should attend to the genetic background of the transgenic animals under study to ascertain that proper controls for strain differences are used. Another limitation of the interpretation of behavioral data from knockout mice is the possibility that compensatory or redundant mechanisms might be activated when a gene is missing. For example, mice lacking the gene for the neuronal isoform of nitric oxide synthase (nNOS–/–) have a 20% increase in the expression of the endothelial isoform of nitric oxide synthase (Burnett et al., 1997). A compensatory mechanism may spare behavioral function and cloud interpretation of the normal contribution of the gene to behavior. Knockout mice are almost always raised by their natural mothers, which are missing one or more genes that may directly or indirectly affect maternal behavior. Thus, any changes in behavior observed in the knockout offspring may reflect the absence of the missing gene or reflect alterations in maternal care. Cross-fostering of matched-size litters can be used to untangle these influences.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

The availability of “inducible” or “conditional” knockouts, in which a specific gene can be inactivated at any point during development or inactivated only in tissue-specific cells, should provide an important tool to bypass the problem of developmental interactions (Holmes, 2001; Nelson, 1997).

Sensorimotor Tests

Some knockout mice have sensory or perceptual deficiencies that can confound interpretations of altered complex behaviors, such as learning, parenting, mating, or aggression (e.g., (Cases et al., 1995; Young et al., 2002). For example, genetically altered mice may suffer from retinal degeneration and fail to perform adequately in tasks such as use of a Morris water maze, which often requires that animals use extramaze visual cues (Hengemihle et al., 1999).

Vision is assessed with a variety of tests, including the visual-placement test and the visual cliff (Zhong et al., 1996). Auditory abilities are assessed with either a clicker-orientation test or an acoustic-startle test (Crawley et al., 2000; Jero et al., 2001; Weisenburger, 2001). Olfactory ability is determined by how long it takes an animal to discover highly odoriferous food (such as cookies, peanut butter, bacon, or cheese) hidden beneath the cage bedding (Nelson et al., 1995) or by odor-discrimination tests (Sundberg et al., 1982). Pain sensitivity can be tested with paw removal from a hot plate or a tail-flick test (Rubinstein et al., 1996); the motor skills of transgenic mice should be assessed before this test to avoid tissue damage caused by slow reaction, rather than high pain thresholds, but in any case, the stimulus should always be terminated after a predetermined time interval selected to avoid tissue damage. The proposed procedures for assessing general motor skills in transgenic mice before behavioral testing and the criteria for early removal of an animal from a potentially painful or distressful stimulus should be described in detail in the animal-use protocol.

Motor Tests

After assessment of sensory abilities, motor abilities and coordination should be assessed. Many strains of mice (such as waltzers, weavers, and staggerers) suffer from movement difficulties that could affect locomotion, coordination, or grooming (Brown et al., 2000). Such motor deficiencies could also affect performance in any behavioral assessment that requires movement (such as depressing a lever or running a maze) or performance of specific behaviors (such as aggression, mating, or parenting). Many transgenic mice display movement or gait disorders (for example, dopamine 1A receptor–/– and GM2/GD2 synthase–/– mice). For instance, mice that are engineered to lack a key enzyme in complex ganglioside biosynthesis (GM2/GD2 synthase) and that express only the simple ganglioside molecular species GM3 and GD3 develop substantial and progressive behavioral neuropathies, including deficits in reflexes, strength, coordina-

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

tion, and balance (Chiavegatto et al., 2000). Quantitative tests of motor abilities determined at the ages of 8 and 12 months revealed progressive gait disorders in complex-ganglioside knockout mice compared with controls, including reduced length and width of stride, increased hindpaw print length, and marked reduction in rearing. Compared with controls, null mutant mice tended to walk in small labored movements and performed poorly in many tasks that required coordinated movements (Chiavegatto et al., 2000).

Assessment of Anxiety

Genetically altered animals may differ from wild-type animals in their emotional responses (fear, anxiety, and defensive reactions). Atypical emotional reactions interfere with responses in learning and memory tasks and with assessment of mating, parenting, or aggressive behaviors.

Several assays of anxiety-like behaviors have been developed. The most common are the so-called exploration-based tests (Holmes, 2001). The premise of these tests is that for some species such as rodents, the innate drive to explore a novel place will be inhibited as aversion to the new space increases. A simple version is the open-field test. High levels of exploration of the open, brightly illuminated area of an enclosure are interpreted as low-anxiety behavior (reviewed by Holmes, 2001). Highly anxious mice stay near the wall of the enclosure. Administration and scoring of this test have been automated, and several commercial products for performing the test are available. Defecation constitutes an additional measure of anxiety; high rates of bolus production are correlated with anxiety in wild-type rodents. Treatment with anxiolytics increases the time spent in the “open” portion of the open field and reduces the number of boluses produced (Holmes, 2001). Obviously, gene manipulations that affect metabolism or food intake could affect bolus production and confound the assay of anxiety. Other exploration-based tests of anxiety include the elevated plus maze, the light–dark exploration test, the emergence test, and the free-exploration test (Belzung and Griebel, 2001; Pare et al., 2001).

The elevated plus maze has become the most commonly used screen for novel anxiolytics, as well as a probe for anxiety in transgenic mice (Holmes, 2001). The elevated plus maze is shaped like a plus sign, has two open arms and two enclosed arms, and is usually raised at least 1 meter above the floor (Lister, 1987). The test animal is placed in the open center of the plus maze, and the number of entries into the closed arms is compared with the number of entries into the open arms over some period (commonly 5–15 minutes). High levels of anxiety correlate with more time spent in the enclosed arms.

The light–dark exploration test is based on rodents’ innate preference for small, dark, enclosed spaces over large, light, open spaces and their innate tendency to explore novel environments (Crawley, 1981). Spending more time in the light side of a box than in the dark side indicates low anxiety.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

All the exploration-based tests of anxiety rely on movement of an animal in a test apparatus. As noted, many genetically modified animals have motor difficulties. The motor deficits must be taken into account in evaluating anxiety. For example, a common assay for fear is freezing in response to a loud noise (startle response). If a transgenic mouse being tested is slow to move, it may appear to be “freezing” for longer periods than its wild-type cohorts and thus appear to demonstrate a high level of fear. The startle response can be modified by classical conditioning, but it also requires “normal” motor abilities. Only after health problems, sensorimotor deficits, and atypical emotional responses in genetically modified animals are ruled out should behavioral assessment proceed. Rodgers (2001) reviews methodologic pitfalls that should be considered by investigators seeking to characterize genes related to anxiety.

Animal Care and Use Concerns

Animal-care personnel are likely to discover behavioral deficits (such as lack of feeding or maternal care) and should be trained to recognize them. Neuroscientists should be aware of potential problems with animals exposed to novel drugs or neurotoxins and with transgenic animals before their development, housing, breeding, or experimental use. Because of potential sensorimotor deficits or general frailty, transgenic mice should be monitored at least once a day by trained observers until the limitations of the animals are well characterized. That concern is especially appropriate when using tasks—such as those involving water immersion, roto-rods, elevated platforms, treadmills, or other mechanical devices— in which there is a high risk of injury to impaired animals. In animals showing specific sensorimotor deficits caused by a neurotoxin or genetic manipulation, it may be necessary to choose tasks or modify tasks so that they do not impose demands beyond the animal’s reduced abilities. For example, the circular platform maze or radial arm maze described earlier will be better choices than a water maze for testing cognitive function in mice with severe motor impairments that interfere with swimming. Because screening procedures often involve testing a given animal in multiple tasks, excellent record keeping practices are imperative. For all the behavioral phenotyping assessments, clear end points (both temporal and performance) for removing animals from the protocol must be identified. Given the unique deficits that may arise from exposure to novel drugs, neurotoxins, or genetic manipulations, it may be necessary to develop different endpoints for the removal of experimental animals than for normal control or wild type animals. Proposed procedures for dealing with each of those issues should be described in detail in the animal-use protocol, and they should be carefully reviewed by the IACUC. As suggested previously, in situations where the consequences of an experimental manipulation are uncertain or unknown, IACUCs are well advised to work with investigators to develop a plan for careful monitoring and periodic re-evaluation to ensure the health and well-being of the animals.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

NEUROPHYSIOLOGIC RECORDING IN AWAKE, BEHAVING ANIMALS

This section discusses some of the issues to consider in preparing and maintaining animals that are used in neurophysiologic recording experiments while they are awake and performing a behavioral task. Technical issues and animal care and use issues are discussed at length in Chapter 4 (“Neurophysiology Studies”).

Because animals are awake during experimental sessions, many of the concerns associated with studies on anesthetized animals are avoided. However, specific considerations are warranted in these studies. Researchers invest a considerable amount of time and effort in the behavioral training and surgical preparation of each animal. As noted in Preparation and Maintenance of Higher Mammals during Neuroscience Experiments (NIH, 1991), the extensive training and surgical preparation, as well as the often long-term experimental use of the animal, presents a number of issues that require the use of professional judgment and flexibility in interpreting the recommendations of the Guide. Adoption of a team approach to these types of studies is essential to ensuring animal well-being and the acquisition of the necessary data.

The study of many important neuroscience questions requires the use of an awake, behaving animal. Behaving preparations make it possible to study cognitive and integrative brain processes by engaging an animal’s active participation. Trained animals can serve as subjects in experiments directed toward understanding of the neurophysiological or psychophysical processes that underlie motor control, sensorimotor integration, learning, memory, and perception (NIH, 2002). The behavioral repertoires of many mammals resemble those of humans, so data generated on awake, behaving animals can be expected to have considerable relevance when extrapolated to human behavior and neurophysiology (NIH, 1991). An understanding of the species-typical behavior of the animals used in awake, behaving experiments is critical for adequately assessing the animal for signs of stress/discomfort/frustration that may be minimized either through an earlier endpoint determination or by modifying experimental procedures or study-related equipment. Additionally, such knowledge will assist personnel in avoiding the use of inappropriate visual cues (for example, a direct stare at a macaque or large hand/arm gestures) that the animal might perceive as threatening or stress-inducing.

Behavioral Training

Experiments on awake, behaving animals generally occur in several stages. Usually, an animal learns to perform a task reliably during an initial training phase. Concurrently with or immediately after this training phase, various devices that are required to quantify behavioral variables, such as eye coils to

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

monitor eye position (Fuchs and Robinson, 1966) or the neurophysiologic consequences of task performance, such as electromyographic electrodes to record muscle activity (Loeb and Gans, 1986) are surgically implanted. (Some of the devices that are typically implanted are described in greater detail below.) After implantation of necessary hardware, data are collected in regularly scheduled recording sessions over a period that can extend for months or years. The use of these animals for this long a period of time makes it even more incumbent upon research personnel to understand the effect of their behavior on that of the animals with which they work. For example, Bayne et al. (1993) demonstrated that positive interaction with nonhuman primates can lead to significant reductions in the expression of abnormal behavior, while Line et al. (1989) have shown that even routine husbandry procedures performed by familiar staff can influence an animal’s physiology, such as heart rate. The potential impact animal care staff can have on animal well-being has recently been reviewed (Bayne, 2002). Indeed, it has been recommended that “a genuine caring attitude” prevail among animal-care staff.

Experiments with behaving animals may involve training animals to perform a specific behavioral task. That allows the neuroscientist to repeatedly elicit and monitor a stereotyped movement, to present sensory stimuli under highly controlled conditions, and to obtain psychophysical discriminations from the animals. In addition, providing animals with a challenging and rewarding behavioral task can stimulate their cooperation in the experiment, reduce their boredom, and generally facilitate collection of high-quality data (NIH, 1991). If the goal of an experiment is to examine variables associated with learning, naive animals are often studied as they learn a new behavior (Lemon, 1984c). That allows the behavioral or neurophysiologic variables that change as a skill is acquired or refined to be compared and measured.

Various training methods and tasks are suitable for achieving experimental goals. In most neuroscience experiments on awake, behaving animals, traditional operant-conditioning paradigms are used. These paradigms require an animal to respond behaviorally to a stimulus to achieve a desired consequence. The most common procedures used in neuroscience experiments are appetitive or aversive (see above, “Use of Appetitive and Aversive Stimuli”). Aversive procedures generally involve exposing animals to some form of noxious stimulus (such as mild electric shock, a bitter food, or an unpleasant sound) when they make an incorrect behavioral response. For some experiments, aversive procedures may be the most appropriate means of training animals to perform a task, because they yield highly reliable behavior with smaller differences between individuals (Toth and Gardiner, 2000). Aversive procedures may also be less likely to upset basic metabolic functions than appetitive procedures. However, it is generally recommended that the use of such aversive procedures conform with Principle IV of the US Government Principles (IRAC, 1985), which states, “Unless the contrary is established, investigators should consider that procedures that cause pain and

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

distress in human beings may cause pain or distress in other animals.” Thus, the level of aversive stimulation applied to an animal generally should not exceed that tolerated by a human being. Not uncommonly, a member of the IACUC will experience the aversive stimulus for him/herself to better understand what the animal will undergo.

Restriction of access to food or water is often used in behavioral neuroscience and neurophysiologic recording paradigms to motivate animals to execute desired behavioral tasks. This process merits attention to specific considerations that are addressed in Chapter 3 (“Food and Water Regulation”). In brief, investigators should provide a sound rationale for using appetitive or an aversive procedures. If access to liquid or solid food is to be restricted, the proposed level of dietary control should be justified, and appropriate monitoring and record keeping procedures should be described in the animal-use protocol. The procedures can be based on the literature or on an investigator’s own experience and should include criteria for determining intervention endpoints for removal of an animal from a particular conditioning paradigm. The goal of the monitoring procedures is not only to keep animals in a highly motivated state but also to maintain their health and welfare. Accordingly, records often include details regarding the animal’s performance on the behavioral task and various physiologic indexes.

Documentation and Record Keeping

Because of the potential health implications of food or fluid restriction, the health status of animals used should be well documented if food or water availability is restricted. Representative animal records might include weight or assessment of hydration status, general appearance or disposition, performance during the behavioral-task session, volume of fluid consumed (earned plus supplemented), dietary supplements or treats that were given, and experimental manipulations that were performed or treatments that were administered. For some species, the welfare of the animal may be further assured by monitoring its behavior in the home cage. Investigators should weigh animals according to suggested guidelines (NIH, 2002) and be alert to changes in mood, behavior, or appearance that indicate a potential medical concern. Individual animals may respond adversely to the weighing process; in such cases, judicious adjustment of weighing frequency or modifying the means of obtaining weights to better accommodate the individual animal may be necessary, and alternative methods of monitoring hydration may be advisable, for example, skin turgor, moistness of feces, and general appearance and demeanor (see “Methods for Assessment of Proper Nutrition and Hydration” in Chapter 3).

There is some likelihood of weight loss during different phases of training (NIH, 2002). An animal’s use in a food- or fluid-restricted behavioral experiment should be assessed with veterinary input if persistent weight loss occurs. No

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

single physiologic measurement will always provide a reliable index of an animal’s well-being throughout the course of a behavioral neurophysiology experiment, but regular monitoring of several measurements (such as of food and water intake, weight, urine and feces, fur and skin, and behavior) usually permits adequate noninvasive evaluation. Each animal has different needs for food and fluids, so flexible criteria are preferable to rigid prescriptions of how much food or fluid animals should receive daily.

Because different animals respond to food or fluid restriction challenges with different physiologic and behavioral accommodations, monitoring of each animal is essential, and adjustment of the restriction protocol is sometimes necessary (Toth and Gardiner, 2000). That is especially important during the initial stages of learning a new behavioral task. Emphasizing the role of professional judgment in these types of experiments, Toth and Gardiner (2000) recommend that:

If task performance is not adequately supporting minimal intakes, the experimenter should re-evaluate and perhaps simplify the training strategy to facilitate the animal’s ability to learn and master the task.

Standard clinical tests will reveal serious pathologic conditions, but the more insidious, gradual deterioration of an animal’s status can be recognized and treated only if there is regular observation and the implementation of professional judgment. Perhaps the greatest challenge in the maintenance of awake, behaving animals is the determination of their overall status. An animal’s overall behavior in its cage is a sensitive indicator of its psychologic and physical status (NIH, 1991). Investigators, veterinary personnel, and when available, behavior experts share in the responsibility of observing behavior, general appearance, and demeanor throughout an experimental regimen. Handlers of animals that are used in behavioral experiments should be knowledgeable and skilled in the interpretation of behavior such that changes that could indicate underlying health and well-being problems are readily identified and reported (Bayne, 2000). To this end, each animal should serve as its own behavior control, with baseline observations made prior to the initiation of the study.

MOOD-DISORDER MODELS

There has been considerable debate about the validity of animal models of human affective disorders. At a minimum, a good animal model of an affective disorder should meet many or all of the following criteria (Redei et al., 2001): strong behavioral similarities with the human disorder, a cause similar to the cause of the human disorder, similar pathophysiology, and similar treatments. Several animal models of affective disorder have been developed, especially for depression. In these models, depressive behavior may be caused by genetic manipulation, environmental perturbations or stressors, or drug treatments (Redei et

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

al., 2001). As is the case in all behavioral research, care must be taken to assess performance in these model systems in a valid and reproducible manner.

Depression

The so-called Porsolt swim test is the most commonly used test for assessment of depression in animal models (Porsolt, 2000; Porsolt et al., 1977). Other tests and procedures include the tail-suspension test, anhedonia (such as with consumption of sucrose solution), learned helplessness, chronic mild stress, olfactory bulbectomy, differential reinforcement of low rate of responding behavior, and conditioned place preference (Porsolt, 2000; Redei et al., 2001; Vaugeois et al., 1997; Willner, 1997). In all those tests, treatment with antidepressants that are effective in treating humans with depression reverses the depressed behavioral responses. It is generally accepted, however, that the Porsolt swim test (behavioral despair) and tail-suspension tests model human depression most closely (Crawley, 2000; Porsolt, 2000).

In the Porsolt test, rodents are placed in a container of water at least 30 cm deep (to prevent an animal from touching the bottom of the container with their tail) and at least 15 cm from the top of the container (to prevent escape). To avoid temperature-related stress responses, the water temperature should be 24–30°C. Rodents placed in water generally swim, but if manipulated with some drugs or brain lesions, they stop swimming and float. Floating is considered a measure of depression because the animals appear to stop trying (learned helplessness or behavioral despair) and because drugs that are effective antidepressants in humans decrease floating time (Crawley, 1999). Genetically modified animals may require special attention; any rodent that fails to swim or float should be removed from the water immediately. However, even if transgenic animals can remain afloat, locomotor difficulties can interact with performance in the Porsolt swim test and cause nondepressed transgenic animals to appear depressed.

The tail-suspension test avoids the problems of locomotion somewhat and avoids the hypothermia and stress associated with forced swimming (Vaugeois et al., 1997). Animals are suspended by their tails and the amount of “immobility” is measured by a force-strain gauge that records all their movements (Steru et al., 1985). Longer periods of immobility are associated with higher depressive scores. The immobility can be reversed with antidepressant treatment (Vaugeois et al., 1997).

Reduced ingestion of a sucrose solution is another reliable indicator of depression-like-behavior in rodents (Gittos and Papp, 2001; Stock et al., 2000). This test avoids some of the problems of locomotion and coordination of the Porsolt test, but if the targeted gene affects metabolism or food intake, its reliability for depressed behavior may be impaired.

In all those behavioral tests of depression, proposed procedures for monitoring, record keeping, and humane intervention should be described in the associated animal-use protocol and approved by the IACUC.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Anxiety

Assessment of anxiety was described earlier in this chapter (“Behavioral Screening of Genetically Modified Animals, Assessment of Anxiety”).

Alcohol and Drug Addiction

Several testing paradigms assess responsiveness of rodents to drugs that have abuse potential, including paradigms involving self-administration of alcohol, cocaine, morphine, or nicotine (Crawley, 1999; Grahame and Cunningham, 1995). Self-administration is typically achieved by requiring the rodent to press a lever or display a place preference. Tolerance, dependence, and withdrawal symptoms can be studied. With this approach, transgenic mice may have locomotor or coordination difficulties that interfere with self-administration (McClearn and Vandenbergh, 2000). Additional information is provided in Chapter 8 (“Addictive Agents”).

Animal Care and Use Concerns

The primary goal of the preceding behavioral assays is the induction of stress or aversive states. It is important for the investigator to determine the earliest possible or least severe endpoint when the manipulation has adverse effects on an animal. Any behavioral test that subjects animals to water has the potential for evoking a stress response. Therefore, it is important that the time that the animal is in the water be minimized and that animals be monitored closely to avoid unnecessary stress. Animals should be dried thoroughly after the swim test, and it is advisable to place their cages on a heating pad for several minutes. The use of unregulated heating pads or heat lamps should be avoided as they can develop hot spots and cause thermal burns.

Continuous monitoring is also important for automated tasks, such as tasks that use roto-rods, platforms, or other devices in which animals may be injured. Because of the likelihood of multiple testing, excellent record keeping is imperative.

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

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

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

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

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.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

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.,

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

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).

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

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

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

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 deter-

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×

mining 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.

Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 123
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 124
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 125
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 126
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 127
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 128
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 129
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 130
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 131
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 132
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 133
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 134
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 135
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 136
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 137
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 138
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 139
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 140
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 141
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 142
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 143
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 144
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 145
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 146
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 147
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 148
Suggested Citation:"9 Behavioral Studies." National Research Council. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press. doi: 10.17226/10732.
×
Page 149
Next: References »
Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research Get This Book
×
Buy Paperback | $39.95 Buy Ebook | $31.99
MyNAP members save 10% online.
Login or Register to save!
Download Free PDF

Expanding on the National Research Council’s Guide for the Care and Use of Laboratory Animals, this book deals specifically with mammals in neuroscience and behavioral research laboratories. It offers flexible guidelines for the care of these animals, and guidance on adapting these guidelines to various situations without hindering the research process. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research offers a more in-depth treatment of concerns specific to these disciplines than any previous guide on animal care and use. It treats on such important subjects as:

  • The important role that the researcher and veterinarian play in developing animal protocols.
  • Methods for assessing and ensuring an animal’s well-being.
  • General animal-care elements as they apply to neuroscience and behavioral research, and common animal welfare challenges this research can pose.

The use of professional judgment and careful interpretation of regulations and guidelines to develop performance standards ensuring animal well-being and high-quality research. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research treats the development and evaluation of animal-use protocols as a decision-making process, not just a decision. To this end, it presents the most current, in-depth information about the best practices for animal care and use, as they pertain to the intricacies of neuroscience and behavioral research.

  1. ×

    Welcome to OpenBook!

    You're looking at OpenBook, NAP.edu's online reading room since 1999. Based on feedback from you, our users, we've made some improvements that make it easier than ever to read thousands of publications on our website.

    Do you want to take a quick tour of the OpenBook's features?

    No Thanks Take a Tour »
  2. ×

    Show this book's table of contents, where you can jump to any chapter by name.

    « Back Next »
  3. ×

    ...or use these buttons to go back to the previous chapter or skip to the next one.

    « Back Next »
  4. ×

    Jump up to the previous page or down to the next one. Also, you can type in a page number and press Enter to go directly to that page in the book.

    « Back Next »
  5. ×

    Switch between the Original Pages, where you can read the report as it appeared in print, and Text Pages for the web version, where you can highlight and search the text.

    « Back Next »
  6. ×

    To search the entire text of this book, type in your search term here and press Enter.

    « Back Next »
  7. ×

    Share a link to this book page on your preferred social network or via email.

    « Back Next »
  8. ×

    View our suggested citation for this chapter.

    « Back Next »
  9. ×

    Ready to take your reading offline? Click here to buy this book in print or download it as a free PDF, if available.

    « Back Next »
Stay Connected!