Testing - Behavioral Measures of Neurotoxicity.pdf

David H. Overstreet and Elaine L. Bailey

There is a wealth of evidence from clinical and epidemiological research to document the fact that a wide variety of exogenous chemicals are capable of producing dementia. Dementia involves declines in learning memory and other cognitive processes, (e.g., problem solving), all of which are necessary for normal adaptations to changing physical and psychosocial environments. The development of animal models of dementia is making it possible to supplement knowledge gained from clinical and epidemiological approaches with information obtained by experimental manipulations of the variables involved.

The material that follows, briefly reviews the research designs, procedures, and specific paradigms used in such experimental studies.

ANIMAL MODELS OF DEMENTIA

With the increasing recognition of the significance to individuals, and to society generally, of the primary degenerative dementias, interest in the development of animal models of dementia has been growing rapidly. Although some skepticism has been expressed about the possibility of constructing such models, a generally optimistic view has prevailed (Heise, 1984; Overstreet and Russell, 1984). For example, several investigators have used neurotoxins such as ibotenic acid, an excitotoxic amino acid, or AF64A, a putatively specific cholinergic neurotoxin, as tools for creating morphological lesions in the central nervous system (CNS), thereby producing cholinergic deficits analo

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gous to those characteristic of Alzheimer's disease (Bailey et al., 1986; Hepler et al., 1985). There have been approaches using neurochemical methods (see chapters by Michalek and Pintor, and Russell). These studies have routinely shown that treatments which interfere with normal cholinergic neurotransmission lead to disruption in measures of learning and memory, thereby providing support for the "cholinergic hypothesis of memory" (Bartus et al., 1982; Coyle et al., 1983). There is also a growing appreciation that the behavioral measures used in these studies are applicable to the detection of the neurobehavioral effects of suspected environmental toxicants (e.g., Walsh and Chrobak, 1987).

Learning and memory are theoretical constructs that cannot be measured directly. They are inferred from observations of behavior under certain specified conditions. Learning is manifested by systematic changes in behavior as a consequence of repeated exposures to the same stimulus environment;

memory, as the preservation of learned behavior over time (Heise, 1984). Any manipulation of an animal's performance may confound the interpretation of the possible effects of that manipulation on learning. As a consequence, an investigator studying animal models of dementia should examine a range of tasks from which learning and memory measures may be extracted, as well as observing, for comparison, other behavioral parameters that are definitely not related to learning and memory. For example, if, during the course of an experiment, food or water is used as a reinforcer, the investigator should measure the effect of the experimental manipulation on the food or water directly. Without elaborating further, it is clear that in order to specify toxic effects on cognitive processes, it is desirable to use batteries of measures rather than to depend upon single assays.

Requirements that animal models of dementia must meet and the characteristics of research designs in which they are put to work have been discussed in detail elsewhere (Heise, 1984; Hepler et al., 1985; Kennett et al., 1987; Olton, 1983; Overstreet and Russell, 1984; Russell and Overstreet, 1984;

Tilson and Mitchell, 1984; Willner, 1984). They are mentioned here only very briefly as a general setting within which to project the more detailed discussion to follow. Specifications for the development of animal models and for safeguards in the use of them have been established by international organizations (e.g., World Health Organization, 1975), by national scientific bodies (e.g., Xintaris et al., 1974), and by individual investigators (e.g., Weiss and Laties, 1975). These specifications include systematic manipulation of independent variables, while eliminating effects of other potentially confounding factors; precise measurement of dependent variables by

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using reliable measuring instruments; attention to the validity of animal models for generalizations to other species, including human; and strict adherence to today's scientific ethics in the care and treatment of animal subjects. With these general points in mind, the use of environmental or pharmacological

"challenges" as methodologies in neurobehavioral toxicology is considered.

ENVIRONMENTAL CHALLENGES

MacPhail et al. (1983) have given several examples of the use of an environmental challenge to uncover a debilitating effect of an environmental toxin. They define environmental challenges as "variables that are either known or suspected to affect a baseline of behavior." In effect, the various tasks described above for measuring learning and memory are environmental challenges because they place some demand on the organism. It is precisely for this reason that they may be likely to reveal some effect of the environmental toxin, whereas standard neurobehavioral toxicology testing would not. It is also possible to infer that a limited number of brain regions might be affected if the treatment results in a disruption of memory.

Among other environmental challenges that can be used in an attempt to uncover an effect of a suspected environmental toxin are manipulations of schedule-controlled behavior (MacPhail et al., 1983). As indicated above, spatial or temporal alternation tasks can be used to infer the working memory of an animal. Some years ago, we reported that rats treated chronically with the anticholinesterase diisopropyl fluorophosphate (DFP) did not develop tolerance to its effects on alternation behavior (Overstreet et al., 1974). This finding correlates with the observation of memory disturbances in humans exposed to organophosphate pesticides (Russell and Overstreet, 1987).

A final group of environmental challenges that might be considered for studying neurobehavioral toxicology are paradigms involving stress. As far as we know, no investigator has used this approach as yet, although it has been widely used on animal models of depression (e.g., Willner, 1984) and to study the effects of some drugs (e.g., Weiss et al., 1961). Among the possibilities are the forced swim test (Porsolt, 1982), the inescapable shock ("learned helplessness") paradigm (Maier, 1984), and restraint (e.g., Kennett et al., 1987).

After the animals are exposed to these various forms of stress (sometimes, during exposure), measures of their ability to move are taken. It is reasonable to hypothesize that animals exposed to environmental toxicants would be more susceptible to these stressful conditions and would exhibit greater reductions in activity than control animals. This approach has been

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used to differentiate between two lines of rats that have been selectively bred for varying responses to the anticholinesterase DFP (Overstreet, 1986;

Overstreet et al., 1988b).

PHARMACOLOGICAL CHALLENGES

The use of pharmacological challenges to uncover changes in an organism chronically exposed to chemical agents is a recent development in neurobehavioral toxicology testing (see Zenick, 1983); as far as we know, this design has been used only rarely in studies of animal models of dementia.

However, the principles underlying these challenges were well known to some investigators much earlier. In our early work on the development of tolerance to DFP, for example, we showed that rats could "become tolerant without acute behavioral changes" (Chippendale et al., 1972). These rats, which received daily low doses of DFP, had reductions in brain acetylcholinesterase activity comparable to other rats treated with higher dosages and showed comparable increased sensitivity to the muscarinic antagonist scopolamine (Chippendale et al., 1972).

In a subsequent study using a daily, low-dose paradigm of DFP treatment and a challenge design, it was found that rats developed subsensitivity to muscarinic agonists within five days of starting treatment. The subsensitivity was complete within nine days, at about the time brain acetylcholinesterase activity was at its lowest (Overstreet, 1974). In still another study using the challenge design, we determined that the muscarinic subsensitivity which follows anticholinesterase treatment can be observed after a single, acute treatment; it first appears at about 48 hours and lasts for about two weeks (Overstreet et al., 1977). In fact, it was these challenge studies which led to the notion that decreases in muscarinic receptor concentrations might be a primary mechanism underlying tolerance development to anticholinesterases (Russell and Overstreet, 1987; Schiller, 1979). Zenick (1983) also called attention to this advantage of the challenge design: by using appropriate challenge agents, some hint of the adapting changes taking place in the central nervous system can be obtained.

The challenge design has also been very useful in understanding the changes that have occurred in our two selectively bred rat lines—the Flinders Sensitive Line (FSL) and the Flinders Resistant Line (FRL). These rats were selectively bred to differ in their responses to DFP (Overstreet et al., 1979).

Subsequently, it was found that the FSL rats were more sensitive to muscarinic agonists (Overstreet and Russell, 1982), which correlated with increased concentrations of muscarinic receptors in the hippocampus and striatum (Overstreet et al., 1984).

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More recently, it has been found that the FSL and FRL rats differ in their sensitivity to agents acting upon other neurotransmitter receptors (Russell and Overstreet, 1987; see Overstreet et al., 1988b, for reviews). Thus, selective breeding for differences in sensitivity to anticholinesterases has led to changes in sensitivity to a range of other drugs. These findings suggest that investigators should challenge their treated animals with a range of compounds; otherwise, they may make conclusions that are not accurate (i.e., a neurobehavioral toxicant may induce adaptive changes in a number of neurochemical systems).

As far as we know, the challenge approach has not been used with much purpose by investigators studying animal models of dementia, even though it is reasonable to expect adaptive changes in the lesioned animals (Finger, 1978).

We would like to describe some of our recent work in which the challenge design has been very useful in exploring the time-dependent changes that occur in rats after hippocampal administration of the neurotoxin AF64A. The challenge approach was used to help answer the question whether AF64A has both pre-and postsynaptic effects at cholinergic synapses because the suggestion has been made that it works mainly presynaptically (Hanin, 1984).

If AF64A destroys cholinergic axons in the hippocampus, one would expect a supersensitivity to develop as an adaptation to the lost cholinergic input. We approached this question by challenging the rats with scopolamine, a muscarinic antagonist, and oxotremorine, a muscarinic agonist, and measuring locomotor activity by direct observation of line crossing in a open field chamber. We were surprised by the initial results, carried out three months after surgery, which showed the AF64A-treated rats to be subsensitive to oxotremorine and supersensitive to scopolamine. These data are consistent with receptor decreases, not increases. Intrigued by these results, we sacrificed the rats and carried out receptor binding assays on the hippocampal homogenates.

There was a significant 30 percent reduction in the number of muscarinic receptors in the AF64A-treated rats (Schiller et al., 1990).

In a subsequent experiment we decided to challenge the rats much sooner after the hippocampal administration of AF64A. At three weeks after treatment, the rats were subsensitive to scopolamine, the antagonist, and supersensitive to oxotremorine, the agonist, which suggests that a supersensitivity had developed.

These animals were then left for several months and rechallenged. At this time they were supersensitive to scopolamine and subsensitive to oxotremorine, confirming our earlier results. Thus, there are time-dependent changes in cholinergic mechanisms in response to hippocampal injections of AF64A. The early changes are consistent with the expected supersensitivity;

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however, a subsensitivity later occurs which is associated with a loss of muscarinic receptors (Schiller et al., 1990).

From this it should be clear that a challenge design may be useful in establishing that changes in sensitivity have occurred as a consequence of some treatment or manipulation. Therefore, information about adaptive changes in animals exposed to neurobehavioral toxicants and the mechanisms underlying these adaptive changes can also be gathered by using similar designs. Zenick (1983) gives a number of examples of how the pharmacological challenge design has been used to uncover an effect of a neurobehavioral toxicant. The use of this design should increase in frequency as more investigators become aware of its utility.

In closing this section, we wish to offer a few words of caution about the pharmacological challenge design. Once one has selected a compound to use, there are still problems about the choice of parameters. Locomotor activity is a useful parameter that is sensitive to a range of compounds, whereas operant responding requires more effort to establish but is more sensitive to drug effects. Another problem is the possibility of choosing only one or a limited range of compounds, which might give the investigators a false picture of the mechanisms underlying the adaptive changes. Whenever possible, a wide range of compounds should be selected. A consequence of multiple compounds is multiple testing; therefore, operant responding is favored over locomotor activity as the dependent variable because it is less subject to shifts in baseline.

MEASURING BEHAVIORAL EFFECTS: SPECIFIC PARADIGMS

It has been said that there is a finite number of measurable behaviors, but that the number is very large. Examination of the research literature on neurobehavioral toxicology indicates that certain paradigms have been favored in studies involving animal models, favored at least in part because of their analogies to human behaviors. The categories in which these paradigms are included is discussed below in some detail.

Inhibitory (Passive) Avoidance

The typical experimental environment in which passive avoidance is generated is a two-compartment box. The animal is placed in the lighted compartment on the first day and given a foot shock upon entering the dark compartment. Memory is inferred from the length

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of time the rat remains in the lighted compartment when put there 24 hours later; the longer the stay, the better is the memory. This task can be a particularly useful one because parameters such as strength of shock, its duration, and the time between testing and retention can be varied to search for differences between groups. Large numbers of animals can also be tested in a short space of time. However, because this task uses aversive stimulation, a number of potentially confounding variables must be checked before a firm conclusion can be reached. For example, a drug (e. g., vasopressin) that has intrinsic aversive properties may appear to enhance the memory of this task.

Similarly, any manipulation that makes the animal more ''fearful'' or alters its sensitivity to shock will influence its performance on the passive avoidance task. These potential effects must therefore be tested by independent means.

Active Avoidance Tasks

There are a number of variations to test environments used in active avoidance testing. Runways and two-compartment boxes with either one-way or two-way avoidance tasks have been used. It is also possible to alter the conditioned stimulus, which is usually either a tone or a light. The basic procedure is to place the animal in the apparatus and, after a brief interval (30 s), give the conditioned stimulus. This stimulus is followed in 5–10 s with the shock, if the animal has not moved beforehand. Thus, measures of both avoidance and escape are recorded. If a treatment has a general debilitating effect on the animals, then both avoidances and escapes should be affected. If the treatment influences learning only, then only avoidances will be affected.

The active avoidance tasks require more effort because most animals require 50 or more trials to reach some criterion of learning. Although the escape measure provides an index of the motor effects of a treatment, there are other problems of interpretation. For example, drugs that stimulate motor activity, such as scopolamine and amphetamine, are known to facilitate active avoidance responding (Barrett et al., 1974). At the same time, scopolamine disrupts passive avoidance performance, and some investigators have used the scopolamine-treated animal as a model for dementia (Flood and Cherkin, 1986).

Another problem with these tasks is that aversive stimuli are used. In conclusion, although active avoidance tasks can be used to measure learning in animals, many other tests must be conducted before other confounding variables can be ruled out.

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