Cognitive Function

Abstract

Many chemicals are neurotoxic, and intoxicated people frequently complain of impaired thinking, paying attention, remembering things, and organizing their lives. Methods for assessing such cognitive functions have been developed in laboratory animals as a means to characterize the effects and determine dose–response relationships for neurotoxic chemicals of concern. Because this work entails application of basic principles of behavior analysis, this chapter begins with definitions and a brief tutorial about the fundamental concepts on which tests of cognitive function are based. We then review and discuss the implementation, benefits, and costs of tests of learning, memory, attention, executive function and impulsivity in mammalian (primarily rodent) models, with examples taken from the neurotoxicology literature. Next, issues and problems of interpretation of behavioral results are discussed, again with examples from the literature. Finally, we discuss the role of testing cognitive function in the context of the new trends in toxicity testing, which focus on high-throughput test methods using simplified cellular and molecular assays.

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Abstract:
Cognition is not a unitary process, but rather encompasses a multitude of functions that are dependent upon overlapping but somewhat separable
nervous system substrates. Exposure to toxic substances in development or adulthood can differentially impact these substrates and their resulting
cognitive functions. The purpose of this article is to introduce and distinguish between broad categories of cognitive functioning, and to showcase
and compare procedures and apparatuses that have been developed to assess the behavioral manifestations of these functions in rodent models, in
normal animals as well as in animals that have been exposed to toxic chemicals. Issues associated with the appropriate, specific, and accurate
interpretation of test results are also discussed.
Keywords: Associative learning; Attention; Conditioning; Executive functioning; Habituation; Inhibitory control; Memory; Motivation; Motor
functioning; Nonassociative learning; Sensitization
Biographical Sketch
Lori Driscoll received her Ph.D. in Biopsychology at Cornell University, where she studied the effects of developmental and adulthood lead
exposure on the cholinergic and GABAergic modulation of learning, attention, and memory in rats. She is currently an Associate Professor of
Psychology and Neuroscience at Colorado College in Colorado Springs, Colorado.
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a0010 Cognitive Function
☆
LL Driscoll, Colorado College, Colorado Springs, CO, United States Q1
ã2017 Elsevier Inc. All rights reserved.
Introduction 1
Definitions 2
Principles of Behavioral Analysis 2
Cognitive Functions: Learning, Memory, Attention and Executive Functions 2
Methods for Assessing Chemical-induced Cognitive Dysfunction 3
Nonassociative Learning 4
Habituation 4
Sensitization 5
Associative Learning 5
Classically conditioned 5
Instrumentally conditioned 6
Issues in the Interpretation of Results 11
Nonassociative vs. Associative Factors 11
Examples of Problems of Interpretation 12
Altered sensory function 12
Altered motor function 12
Task specificity 12
Cognitive Science and Toxicity Screening 13
Summary and Conclusions 13
References 14
s0010 Introduction
p0010 The Q2fact that exposure to chemicals can result in adverse effects on the structure and function of the central and peripheral nervous
system of humans has been documented in numerous review papers (Anger, 1990; Anger and Johnson, 1985; Grandjean and
Landrigan, 2006, 2014), and books (Chang and Slikker, 1995; Gupta, 2006; Harry and Tilson, 2010), and federal guidelines for risk
assessment (U.S. Environmental Protection Agency, 1998). Anger and Johnson (1985) reviewed the toxicity of 750 chemicals that
affect the nervous system, and identified some 120 different effects related to functions of the nervous system associated with
exposure to those chemicals. Manifestation of neurotoxicity in humans may be categorized as chemical-induced alterations in
motor, sensory, affective, personality, and cognitive function. Of these effects, alterations in cognitive function appear to be the
most diverse and include changes in learning, sustained and selective attention, categorization, coding, concept shifting, distract-
ibility, memory, pattern recognition, reading, vocabulary, spatial relations, mathematical abilities, and intelligence. The nature and
extent of alterations can depend on the timing and type of exposure, but effects have been found following exposure to many
environmentally relevant chemicals such as lead, mercury, carbon disulfide, styrene, solvent mixtures, and pesticides (reviewed by
Anger, 2003).
p0015 Moreover, it has become clear that the incidence of neurodevelopmental disabilities is rising, affecting approximately one in six
children under the age of 18, or more than 10 million children in the United States alone (Boyle et al., 2011). Recent reviews
identified a dozen chemicals with clear developmental neurotoxicity and some 200 more with adverse clinical consequences in
adults (Grandjean and Landrigan, 2006, 2014). The societal cost of these developmental effects can be enormous: chemical
exposure during childhood has been estimated to lead to a population-based loss of cognitive capacity, indexed by a reduction in
IQ scores, on par with pre-term birth and attention-deficit disorders (Bellinger, 2012).
p0020 Because chemicals can adversely affect cognitive function in humans, considerable effort has been made to characterize their
effects using animal models. Information from such models will be necessary to: evaluate whether chemicals identified as
potentially neurotoxic by screening methods actually do affect cognitive function; identify and characterize the mechanisms or
pathways by which effects at these targets lead to cognitive dysfunction; address issues of susceptibility and variability, which
require understanding the compensations and interactions that only a whole organism can engage; and improve our understanding
of the neurobiological underpinnings of cognitive function.
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☆
Change History: June 2017. LL Driscoll removed author PJ Bushnell, and changed corresponding author to LL Driscoll. Subheadings for Attention and
Executive Functioning were raised one level to be equivalent to the other major cognitive processes. Small editorial changes made throughout to increase
clarity, and some newer references were added.
This is an update of PJ Bushnell, HA Tilson, Cognitive Function, Comprehensive Toxicology, Second Edition, 2010, Volume 13, Pages 367–387, ISBN
978-0-08-046884-6, 10.1016/B978-0-08-046884-6.01322-1.
Comprehensive Toxicology, 3rd Edition http://dx.doi.org/10.1016/B978-0-12-801238-3.02206-6 1
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p0025 This article has several purposes. First, it provides working definitions of cognitive functions, such as learning, memory and
attention, in terms frequently used by behavioral toxicologists. It is important to have a common vocabulary to assess methods
used in this area of research. Second, it presents an overview of some of the procedures commonly used in behavioral toxicology to
assess the effects of chemicals on cognitive function in animals. It should be noted that this overview is not intended to be
comprehensive or complete, but is intended to illustrate specific points by discussing examples. Finally, this article discusses some
critical experimental and conceptual variables that are important for studies on chemical-induced cognitive dysfunction, and
touches on the potential problems and opportunities for analysis of cognitive function in whole animals in the context of current
efforts to implement simple high-throughput tests to screen chemicals for toxicity.
p0030 Due to space limitations, this article reviews methods only for mammalian models, primarily in rodents. Considerable effort is
underway at present to develop behavioral models of cognitive functions and other processes in non-mammalian species, with the
goals of bridging the gap between simpler cell-based or molecular systems, understanding mechanism of action, probing the
genetic bases both of cognition and susceptibility to toxicants, and increasing the speed and efficiency of chemical testing. Readers
who are interested in these complementary models are referred to reviews of approaches in zebrafish (Levin, 2011; Levin and
Cerutti, 2008; Truong et al., 2014), fruit flies (Kahsai and Zars, 2011; Rand, 2010; van Swinderen, 2011a,b Q3) and C. elegans (Bessa
et al., 2013; Rankin et al., 1990; Sasakura and Mori, 2013).
s0015 Definitions
s0020 Principles of Behavioral Analysis
p0035 Cognition is a psychological term that includes the processes of learning, memory and attention, as well as perception, language,
intelligence, and reasoning. Cognitive phenomena are essentially internal psychological processes which, from the experimental
point of view, must be inferred from overt changes in an organism’s behavior (Bushnell, 1998). Behavior can be defined as what an
animal does or as the observable product of the sensory, motor, and integrative processes in the nervous system (Cory-Slechta et al.,
2001; Eckerman and Bushnell, 1992). Specific behaviors, as measured by performance in one or more behavioral tasks, reflect the
state of functioning of particular neural systems.
p0040 Behavior can be unconditioned (unlearned) or conditioned (modified by learning). Experimental psychologists have further
dichotomized conditioned behavior into two classes: respondent or operant. A respondent behavior is elicited by a specific
identifiable stimulus. There is usually a defined temporal relationship between the stimulus and the elicited response. Examples
of respondent behaviors include kineses, taxes, reflexes, and species-specific behaviors. Operant behaviors are not elicited by a
single, identifiable stimulus, but are emitted or occur voluntarily and are controlled by their consequences. An emitted behavior
that is brought under the control of contingencies becomes an operant behavior; these behaviors include discriminated conditional
responses and schedule-controlled behaviors.
p0045 Respondent and operant behaviors can be modified by conditioning or learning. Respondent learning or conditioning refers to
the repeated pairing of an initially-neutral stimulus with a stimulus that elicits a specific response—i.e., an unconditioned stimulus
(US)—that elicits an unconditioned response (UR). Eventually, the previously neutral stimulus becomes a conditioned stimulus
(CS) that elicits a conditioned response (CR) on its own.
p0050 Operant responses become conditioned by the presentation or withdrawal of stimuli. By convention, the presentation of a
stimulus is termed a positive event (because of the addition of something that was previously absent), and the removal of a
stimulus is termed a negative event (because something that was present is now absent). An operant response is considered to be
reinforced if it occurs more frequently than it did before conditioning, and punished or extinguished if it occurs less frequently.
If the probability of a response increases after the presentation of a stimulus, then that stimulus is defined as a reinforcer and
positive reinforcement has occurred. Examples of positive reinforcers include food, water, and affection. If the probability of a
response increases after a stimulus is removed or terminated, then negative reinforcement has occurred. Stimuli that inhibit
behaviors are punishers. Examples of punishers include pain and threats of harm. Finally, behaviors can be inhibited or
extinguished by removing positive reinforcers. The process of changing behavior by application of reinforcement contingencies
is referred to as operant or instrumental conditioning.
s0025 Cognitive Functions: Learning, Memory, Attention and Executive Functions
p0055 Learning can be defined as “an enduring change in the mechanisms of behavior that results from experience with environmental
events” (Domjan and Burkhard, 1986), or a “relatively permanent change in an organism’s potential for responding that results
from prior experience or practice” (Kimble, 1961, p. 2). Inherent in these definitions are the ideas that learning is something that
occurs internally; it is inferred from changes in behavior or the probability of a behavior; and it is relatively persistent. That is,
learning has a neural basis which can be measured behaviorally, and is more enduring than changes in behavior due to arousal,
fatigue, adaptation, or disease.
p0060 Traditional definitions of memory include many of the same elements associated with learning, that is, internal phenomenon,
behavioral manifestation, change in potentiality, and persistence. However, memory differs from learning in that memory concerns
persistence of a behavioral change after the change has been acquired or learned (Eckerman and Bushnell, 1992). Furthermore,
memory studies indicate that there are clear cases in which knowledge of preceding events can have a transient effect on subsequent
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behavior and that memory can be modified by conditions that do not co-vary directly with learning: for example, retroactive and
proactive interference, stimuli that evoke latent information (reminders), and contextual cues (Kimble, 1961). Studies of memory
have indicated the existence of a capacity-limited or short-term memory, and a rather less limited long-term memory, and have
shown that the neurobiological substrates for these forms differ (Squire, 1986). Long-term memory may further be divided into
memory associated with information based on skills or procedures and information based on specific facts or data. Declarative
memory consists of explicit facts, episodes, lists and routine information. Procedural memory is implicit and accessible only
through performance. Declarative memory can be episodic or working, that is, relative to a specific time and place; or semantic or
reference, that is, facts or data that pertain to a broader range of situations or times. Semantic/reference memory is used to
remember what kind of car you drive, and episodic/working memory is necessary to remember where your parked it today.
Procedural memory includes memory for skills, priming effects, respondent conditioning, habituation, and sensitization.
p0065 Attention refers to a number of hypothetical constructs by which the nervous system apprehends and organizes sensory input
and generates coordinated behavior (James, 1890). Varieties of attention may be dichotomized conveniently into an ability to
sustain attention over time and an ability to attend selectively to specific discriminative stimuli while filtering out other stimuli.
Behavioral tests of sustained attention rely on manipulations of stimuli in the temporal domain; tests of selective attention typically
manipulate spatial, modal, or other non-temporal qualities of the discriminative stimuli that control behavior. Behavioral methods
have been designed either to assess one of these aspects of attention or to enable measuring both, by manipulation of test
parameters (Bushnell, 1998; Bushnell and Strupp, 2009; Driscoll and Strupp, 2015).
p0070 Executive functioning refers to cognitive processing that can be described as top-down, active manipulation of information or
control of behavioral states (Cummings, 1993)(Welsh et al., 1990). Executive functioning deficits are commonly observed in
humans exposed to neurotoxic agents and drugs of abuse, whether the exposure occurs during early development (Mattson et al.,
1999; Noland et al., 2003; Warner et al., 2006) or in adulthood (Ersche et al., 2006). Executive functions are mediated by
overlapping but unique prefrontal cortical circuits (Alvarez and Emory, 2006; Miller and Cohen, 2001). This situation has
implications for toxicological testing, because a given chemical exposure may impact some functions but not others (Driscoll
and Strupp, 2015). Executive functions are also dissociable in rat prefrontal cortex, although the circuitry differs between humans
and rats (Uylings et al., 2003).
p0075 Differences between learning, memory, attention and executive function can be difficult to discern because they do not occur in
isolation: behavior is inevitably the result of simultaneous operation of all of the processes involved. For example, a subject will not
remember a stimulus to which s/he was not attending; past experience (learning and consequent memory) will determine the
stimuli to which s/he attends; and executive control of behavior will affect the way information is manipulated and resulting
behavioral changes are expressed. Similarly, one cannot remember something that has not been learned, nor can learning occur
without memory. Nevertheless, it is possible to design tests that can emphasize specific processes, by appropriate training, control
of the testing environment, and cautious interpretation of the data.
p0080 Finally, given that these hypothetical cognitive processes cannot be measured directly, it is fair to ask whether they can be
modeled in animals at all. To the extent that they can be studied objectively in humans through analysis of non-verbal behavior,
they can also be studied in animals. Further, parallel studies in humans and animals can generate conceptual and procedural links
between the species, to facilitate development of animal models of human attentional processes. However, we caution that a model
is just a heuristic tool for predicting effects in the real world. As such, a model is a useful simplification of a complex system that
may help to understand that system, at the risk of over-generalization and assumption of spurious causal relationships. It is thus
necessary to understand and maintain the links between the model and the system of interest (Bushnell, 1998).
p0085 The advantage of tests of cognitive function is that they capture many of the deficits that are seen in intoxicated humans.
However, training animals to perform these tasks can require many sessions (from days to weeks), thus reducing their attractiveness
to those who require high throughput.
s0030 Methods for Assessing Chemical-induced Cognitive Dysfunction
p0090 As described above, cognitive functions include broad categories of learning, memory, attention, and executive function. These
hypothetical processes can be assessed using measurements of behavior using appropriate experimental procedures. However,
because behavior is the integrated output of all of the processes, procedures do not map uniquely to processes. Furthermore,
because several procedures can be implemented in a given apparatus, depending upon the behavioral contingencies that are
arranged, the mapping of apparatus to procedures and processes is also complex. Table 1 provides examples of procedures and
apparatuses that are often used to assess the cognitive dysfunction. This tabulation is neither complete nor exclusive, but may be
useful for understanding the relationships among processes, procedures, and equipment used for assessing these functions.
p0095 The following section describes methods for testing the functions shown in the left column of the table. However, given the fact
that many procedures can be implemented in most of the apparatuses in the table, and the likelihood that choices of procedure will
often be based on the apparatus available, the section is organized in general by the process of concern, but also by the procedures
and equipment that are commonly used to assess them.
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s0035 Nonassociative Learning
p0100 Nonassociative learning consists of changes in fundamental behaviors (e.g., motor activity, startle reflex, limb withdrawal) that
occur with repeated presentation of environmental stimuli, such as a specific testing environment, or a directed stimulus.
Nonassociative learning can involve both habituation (a decrease in response) and sensitization (an increase in response).
Although these forms of learning do not require active cognitive processes to occur, the changes in the nervous system that
occur as a result of the learning, at least at a physiological level, are similar to those that occur when more complex, explicit forms of
learning take place (for a review of the physiology of nonassociative learning and applications to toxicology, see Rossi, 1996).
s0040 Habituation
p0105 Habituation reflects a reduction in exploration of, or response to, a stimulus or test environment as it becomes familiar to the
animal. Acquiring this familiarity requires the animal to learn and remember the stimulus: thus reduced habituation can be taken
as evidence for impaired learning or memory. Habituation of activity in an open field has been used extensively by Per Eriksson’s
group to explore the effects of perinatal exposure to chemicals on the ontogeny of working memory. For example, Viberg
et al. (2003) exposed young NMRI mice on post-natal day (PND) 10 to 0.45, 0.9, or 9.0 mg/kg of a flame retardant, polybromi-
nated diphenyl ether (PBDE) 153, and tested their motor activity at 2, 4 and 6 months of age. At each age, activity of control mice
decreased to near zero across a 60-min test session. Activity in treated mice was lower than control in the first third of the session
and higher than control in the last third. The authors interpreted this pattern of activity as reflecting impaired habituation. This
group has demonstrated a unique sensitivity of these mice to treatment on PND 10 with a wide variety of toxicants, including PCBs
and methyl mercury (Fischer et al., 2008), polyfluorinated substances (Johansson et al., 2008; Viberg et al., 2013) and bisphenol-A
(Viberg et al., 2011).
p0110 Habituation has also been assessed with novel-object recognition tests, which have been used effectively to study learning and
memory in rodents, monkeys and human infants. In this paradigm, a specific stimulus is presented to the subject, and is
subsequently presented again paired with a novel stimulus. Given the normal tendency to explore novel stimuli and ignore
familiar ones, a memory deficit is inferred if the subject does not explore the novel stimulus in preference to the familiar one.
p0115 Novel-object recognition tasks were developed for animals from studies of the development of intelligence in human infants
(Fagan, 1970), and the “Fagan Test” has been used to estimate the consequences to humans of perinatal exposure to environmental
chemicals (e.g., PCBs, mercury, and lead: (Boucher et al., 2014; Jedrychowski et al., 2008)). The paradigm has also been applied to
studies of the effects of exposure to similar chemicals in infant monkeys (Burbacher and Grant, 2012), and considerable knowledge
has been gained about the neurobiological basis of performance in this task using primate models (Paule et al., 2012). Theory and
technique for use of this approach in rats (Antunes and Biala, 2012; Ennaceur, 2010) and standardized protocols for novel-object
recognition tasks have been published for mice (Leger et al., 2013).
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Table 1t0010 Some examples of procedures used in neurotoxicological research to study cognitive processes in animals
Cognitive process Experimental procedure Apparatus
Nonassociative learning
Habituation Motor activity Photoactometer, Figure-8 Maze
Sensitization Kindled behavioral responses Observation
Associative learning
Classical Flavor aversion
Eye-blink
Trace Fear
Water-bottle test
Conditioning chamber
Conditioning chamber
Instrumental
Negative reinforcement Passive avoidance Light-dark box
Active avoidance Shuttle-box, operant chamber
Water escape Morris and Biel mazes
Other escape Barnes maze
Positive reinforcement Spatial orienting Radial-arm, Morris water maze
Discrimination and reversal Operant chamber, T-Maze, Morris water maze
Repeated acquisition Radial maze, operant chamber, 3-Panel runway
Memory
Working Delayed alternation T-Maze
Delayed matching Operant chamber
Spatial orienting Morris water maze
Reference Avoidance conditioning Light-dark box
Persistence of discrimination learning Operant chamber, Mazes, 3-Panel runway
Attention Signal detection 5-Choice serial reaction time, 2-Choice operant
Executive function Set-shifting Operant chamber, maze, sand-digging
Go no-go Operant chamber
4Cognitive Function
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p0120 In rodent tests, objects are used as stimuli, and are typically presented to an animal in an open arena. Exploration of the objects
is scored visually using behavioral criteria (e.g., frequency and/or duration of approach, contact by the nose or whiskers), and the
relative degree of exploration of the novel object is calculated. For example, Lin et al. (2013) dosed adult C57BL/6 mice with
atrazine at doses of 0, 5, 25, 125, or 250 mg/kg/day for 10 days. On Day 8 of this regimen, a mouse was placed in an open arena for
30 min of acclimation to the arena. On Day 9, it was placed again in the arena, which now contained two identical plastic objects,
for 5 min. Then, after a 1-h period in the home cage, it was placed a third time in the arena, which now contained one of the
original objects paired with a novel one. Exploration of the two objects was quantified by video tracking software. Calculation of a
Novelty Preference Index (NPI) (Sik et al., 2003) showed a dose-related decrease in exploration of the novel object, suggesting a
deficit in recognition memory.
s0045 Sensitization
p0125 Behaviorally, sensitization manifests itself as the opposite of habituation: an initial response to a stimulus becomes exaggerated,
rather than attenuated, with repeated exposure to the stimulus. (This nonassociative form of sensitization is not to be confused with
classically conditioned sensitization; Carew et al., 1981.) However, sensitization is a separate process from habituation and
conditioning, and in fact these three processes can interact, additively or angatonistically, in the context of responses to chemicals
(Bell et al., 1999). Sensitization has been observed in humans (Miller, 1994) and non-human animals (Gilbert, 1995; Robinson
and Berridge, 2000) following an initial exposure or exposures to pesticides, solvents, and psychomotor stimulants. Sensitization
consists of an initiation period, with one high-dose exposure or a series of spaced lower dose exposures, followed by the elicitation
of the sensitized response to either the same substance or different substances (e.g., paint fumes, foods). Initiation can be triggered
by chemical exposures or stressful events (Leao et al., 2012). Similarly, the eliciting substance(s) may or may not themselves be
toxic (Bell et al., 1997). The sensitized behavioral response can vary, from increased neural excitability that can trigger seizures
(a phenomenon known as kindling) to perceptions of unreality and cognitive disruptions in sensitized humans.
p0130 Sensitization may be the physiological phenomenon that underlies at least some cases of mutliple chemical sensitivity (MCS),
in which an individual becomes intolerant to the presence of many different classes of chemcials (Bell et al., 1997). Kindling is used
as an experimental technique, alone and in conjunction with chemical exposures, as an animal model of sensitization and to study
MCS. In rodents, kindling is established by applying low-intensity electrical stimulation to the amygdala one to two times daily
until behavioral seizures of a particular severity and frequency occur (Gilbert, 1995). This pattern of repeated stimulation sensitizes
the neural tissue, making it more susceptible to behavioral seizures. This model can be used to explore the ability of chemical
exposures to modulate kindling, or the interaction of already kindled tissue with toxic exposures. In both cases, appropriate
controls are needed. If exposure occurs during the kindling process, kindling should also be measured in a separate group of
animals not exposed to the toxicant. If exposure occurs after kindling, naı¨ve animals and animals that have been previously kindled
can then be exposed to the chemical of interest to compare sensitivity to a subsequent course of stimulation.
s0050 Associative Learning
p0135 Associative learning or conditioning is usually categorized as being classical (respondent) or instrumental (operant) and is formed
following the association between a stimulus and a behavioral outcome. It is important not to confuse the process being assessed
(e.g., learning) from the procedure used to assess it (e.g., acquisition of a discrimination) (Overmeier, 1987). Processes are
hypothetical cognitive constructs that are inferred from behavior in specific, well-controlled test environments. Procedures are
the operations and manipulations that are used to generate the behavior of interest. It is also important to recognize that the same
process can be assessed by multiple procedures, and that the same procedure can be implemented in more than one test
environment. For example, the process working memory can be assessed by several procedures including delayed alternation,
delayed match-to-sample, and food retrieval behavior in the radial-arm maze. Moreover, delayed alternation and delayed match-
to-sample procedures can be implemented in operant test chambers (“Skinner boxes”), in a variety of maze environments (e.g.,
T-maze, radial-arm maze, or water maze) and, in primates, in the Wisconsin General Test Apparatus. See Bushnell (1998) for a
more thorough discussion of these issues.
s0055 Classically conditioned
s0060 Flavor aversion
p0140 An example of a classical conditioning paradigm is the flavor aversion procedure, which is based on the finding that animals will
avoid consuming solutions with flavors previously paired with illness. Memory for the aversion is assessed by presenting the animal
with the flavor alone. Peele et al. (1989) exposed rats to trimethyltin (TMT) and assessed them 30 days later for conditioned flavor
aversions to saccharin by pairing it with lithium chloride, which induces nausea. Two days after conditioning, the rats’ preference
for the saccharin solution was measured. Rats dosed with TMT did not differ from controls if delays of 0.5 h or 3 h occurred
between saccharin exposure and lithium. After a 6 h delay, however, there was a significant reduction in the aversion to saccharin in
the TMT-exposed rats, suggesting impaired memory for the conditioned association. The biological basis of flavor aversion
conditioning has been elucidated (Miranda, 2012; Sandner, 2004), but this method of assessing the effects of chemicals on
memory has not to our knowledge been pursued.
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s0065 Eye blink
p0145 Classical conditioning of the eye-blink response has been used to evaluate learning. In this method, a CS, such as a tone, is paired
contingently with a US, such as a brief air puff to the eye. The air puff elicits a reflexive eye-blink; after repeated conditioning trials,
the tone comes to elicit the eye-blink response in the absence of the air-puff. Using this procedure, Stanton and Freeman (1994)
reported that developmental exposure to methylazoxymethanol (MAM), an antimitotic agent, interferes with the acquisition of the
eyeblink response in infant rats. MAM is known to affect the development of the cerebellar granule cells, which are essential
components of the cerebellar and brain-stem systems that mediate the eye-blink response.
p0150 The eye-blink conditioning paradigm has several advantages for studies concerning the effects of chemicals on learning. The
neural substrate has been extensively studied; it can be measured in a number of species, including humans; it lends itself to
comparisons across ages; it does not require language competence; and it requires a relatively simple motor response (Stanton and
Freeman, 1994). Further work with this preparation has documented deleterious effects of prenatal exposure to ethanol (Brown
et al., 2007), which has led to use of the method in an animal model of fetal alcohol spectrum disorders (FASD). Thus, experiments
in rats (Murawski et al., 2013) have identified mechanisms likely to mediate the deficits observed in children with FASD (Jacobson
et al., 2011).
s0070 Trace fear
p0155 In fear conditioning methods, an initially-neutral CS (e.g., tone or light) is presented to an animal either coincidentally with an
aversive US (usually electric shock), or preceding the US. If a delay is interposed between the CS and US, conditioning is presumed
to occur to a ‘trace’ of the CS, and the method is thus ‘trace fear conditioning’. Insertion of the delay slows acquisition of the
contingency between the CS and US and also engages the hippocampus, the activity of which is necessary for learning under trace
conditions (Shors et al., 2000). Because rodents tend to suppress their activity (‘freeze’) in response to an aversive stimulus,
assessment of conditioning in rodents typically involves quantifying the animal’s tendency to freeze in response to the CS after it
has been paired with the US.
p0160 An example of this method was reported by Gilbert (2011) in a study of developmental thyroid insufficiency induced by
perinatal administration of n-propylthiouracil (PTU) in rats. The test required two days. On Day 1 the rats experienced paired
presentations of a light-tone CS and foot-shock (US); in one of the two reported experiments, a distractor stimulus was also present
throughout this conditioning phase. The rats suppressed their motor activity during and after the shock. On Day 2, the rats were
returned to the same test box and their activity was measured in the absence of the CS or US. Conditioning to the experimental
context was defined as the degree of suppression of their activity (‘freezing’) relative to that observed the previous day before
conditioning. The rats were then placed in a different chamber, where freezing was measured in response to the CS that had
previously been paired with shock. Conditioning to the cue was defined as the degree of freezing during a 3-min period following
presentation of the CS.
p0165 Results showed that PTU treatment impaired both context and cue learning, but only when the distractor stimuli were presented
during training. In the presence of the distractor, the high-dose group showed reduced freezing (impaired learning) and the
low-dose groups did not differ from controls. Presentation of distractor stimuli during trace fear conditioning increases
the difficulty of the task and appears to engage cortical circuitry associated with attentional processes (Han et al., 2003). Thus
the effects of thyroid insufficiency in this study suggested impairments in both attentional and memory processes.
p0170 In summary, fear conditioning methods have several advantages, including a short training and test period (less than a week,
including acclimation of the animals before the 2-day experiment) and the ability to differentiate among neural components
mediating learning. That is, delay conditioning, in which the US follows the CS without any intervening trace interval, engages the
amygdala but not the hippocampus or cortex. Adding the trace time interval between the CS and US recruits memory processes that
depend upon hippocampal circuitry, and adding distractors in the trace method recruits cortical structures involved with attention.
s0075 Instrumentally conditioned
s0080 Procedures using negative reinforcement
p0175 Negative reinforcement occurs upon termination of an aversive stimulus: that is, when an animal emits a behavior that prevents or
terminates a noxious stimulus, that behavior is said to be negatively reinforced. Note that it is the termination of the noxious
stimulus that is the negative reinforcer, not the stimulus itself.
s0085 Passive avoidance
p0180 One common passive avoidance method involves placing a rat into a chamber divided into two compartments separated by a door.
The two compartments are often of different size and one of them may be lit, while the other is dark. Mundy et al. (1990) infused
colchicine into the nucleus basalis magnocellularis of rats to destroy ascending cholinergic fibers in the cortex. Rats were tested for
effects on cognitive function 14 days after treatment. The rats were placed into a smaller lit compartment for 10 s, after which a
guillotine door was opened. The latency to enter a larger, dark chamber was recorded. Upon entering the dark chamber, the rats
received a mild electric shock through the grid floor. Forty-eight hours later, memory was assessed by repeating the entire
procedure, omitting the shock. Prior treatment with colchicine did not affect the initial latency to cross-over during training.
On the memory test, however, colchicine-treated animals had significantly lower latencies to enter the larger compartment,
suggesting a deficit in memory.
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p0185 The passive avoidance task has been used frequently in neurotoxicological studies to study reference memory (Eckerman and
Bushnell, 1992). For example, the pesticide chlorpyrifos is an organophosphorus inhibitor of cholinesterase, which has been
implicated in disruption of cholinergically-mediated working memory in adult rats exposed prenatally (Icenogle et al., 2004)as
well as in rats dosed as adults (Bushnell et al., 1993). Recent work has shown that prenatal, but not postnatal exposure to
chlorpyrifos impaired passive avoidance in rats (Ricceri et al., 2003; Vatanparast et al., 2013).
s0090 Active avoidance
p0190 In contrast to passive avoidance tasks in which withholding a response is reinforced, active avoidance tasks require that the animal
perform a specific response to avoid or escape an aversive stimulus (e.g., electric shock). Frequently, the onset of the aversive
stimulus is preceded by a warning stimulus that is terminated if a conditioned response is made. If the correct response is not made,
the aversive stimulus is presented for a set period of time or until the correct response is emitted. One-way active avoidance requires
the animal to move in one direction to escape or avoid punishment. In the two-way avoidance task, the animal is required to
shuttle between two compartments to escape or avoid punishment.
p0195 Active avoidance tests have been used to assess the effects of diet on the toxicity of the nerve agent soman (Myers and Langston,
2011), the effects of early postnatal exposure to diesel exhaust particles (Yokota et al., 2011), and the effects of prenatal exposure to
methylmercury (Carratu et al., 2008). In the latter study, pregnant rats were dosed with methylmercury or vehicle on GD 15, and
their 90-day-old male offspring were trained to avoid shock that was signaled by an auditory cue. Acquisition of the conditioned
avoidance response across 100 trials of training was significantly impaired in the mercury-treated rats. Shock sensitivity, motor
activity, and auditory function in siblings of the affected rats were not affected by mercury, supporting the conclusion that learning
was impaired in these animals.
s0095 Water escape: Morris water maze
p0200 The Morris water maze (MWM) is an apparatus that enables a variety of well-established tests of spatial working and reference
memory in rodents, particularly rats (Morris, 1984; Vorhees and Williams, 2006). In the basic procedure, the animal is placed into a
large tank of opaque water and is required to swim to a submerged (i.e., hidden from view) platform in order to escape the water;
therefore, escape is the negative reinforcer. The task taps memory because the animal is first trained to swim to a visible platform.
Once the platform is hidden, the animal must remember its location using only egocentric (i.e., body-centered) or allocentric (i.e.,
external) cues. Swim path length, swim speed and heading direction are measures obtained by human observers or video tracking
methods to assess the animal’s performance. Early studies (Morris, 1984) showed that the rats preferentially use allocentric cues—
typically the walls and configuration of the room that houses the maze—to navigate to the platform.
p0205 This spatial navigation in the MWM is particularly sensitive to alterations in the cholinergic and glutamatergic systems
(McNamara and Skelton, 1993). Different neural systems for memory are used to acquire the task: the use of allocentric cues
requires explicit (i.e., hippocampal-dependent) memory, whereas the use of egocentric cues requires implicit (e.g., striatal-
dependent) memory (McDonald and White, 1993). Other variants of the task can be used to differentially assess amygdalar,
cortical, and cerebellar memory systems (D’Hooge and De Deyn, 2001). The persistence of memory can be measured in the maze
by introducing delays between acquisition and recall, or by probe trials, in which the platform is removed. In a probe trial, intact
memory is indicated by the animal’s tendency to swim in the platform’s former location. Reversal learning can also be assessed by
moving the platform to a different sector of the maze (de Bruin et al., 1994).
p0210 The MWM does not require extended training, in contrast to appetitively motivated spatial memory tasks such as the radial arm
maze (see discussion below). Performance also seems to be relatively robust in the face of motivational or locomotor deficits
(Fitzgerald and Dokla, 1989; Morris et al., 1982; Vorhees and Williams, 2006), which sometimes accompany toxic exposures.
In addition, experiments in humans and rats demonstrate that many MWM procedures translate across species. For example, both
humans (in a virtual MWM) and rats exhibit a preference for egocentric responding when they choose an initial swim path, but they
rely on extra-maze cues as they approach their destination (Hamilton et al., 2009).
p0215 The MWM has become an increasingly popular tool for assessing learning and memory in toxicology. For example, impaired
performance has been reported in rodent models of fetal alcohol syndrome (Richardson et al., 2002; Savage et al., 2002). In one
study, rat offspring of dams who consumed 3% and 5% ethanol liquid diet during pregnancy demonstrated impaired learning of
the position of the submerged platform (measured as change in path length from the first to second trials) when the location of the
platform was changed from day to day, suggesting deficits in working memory. Interestingly, human males with fetal alcohol
syndrome also showed working memory deficits in performance in the virtual MWM, particularly on probe trials (Hamilton et al.,
2003). The MWM has been used to study the effects on learning and memory of many other compounds as well, including
chlorpyrifos (Canadas et al., 2005; Jett et al., 2001), PBDEs (Viberg et al., 2006), disulfoton Q4(Llorens et al., 1993a,b), toluene (von
Euler et al., 1993), and lead (Nihei et al., 2000).
s0100 Water escape: Biel and Cincinnati mazes
p0220 Water mazes of other configurations have also been used in neurotoxicology, including the Biel and Cincinnati mazes (Vorhees,
1987). The Biel maze is a multiple-T maze that requires that the animal make a series of left or right turns before finding the arm
leading to escape from the water in the maze. The Biel maze has been used to study the developmental neurotoxicity of
methylmercury (Vorhees, 1985), and solvents (Cruzan et al., 2005;Faber et al., 2007) among other compounds. The Cincinnati
maze is an expanded version of the Biel maze that has been used extensively to evaluate persistent effects of developmental
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exposure to drugs including phenytoin (Vorhees et al., 1991), thalidomide (Vorhees et al., 2001), and stimulants (Skelton et al.,
2008; Williams et al., 2003). These mazes, in contrast to the Morris water maze, are specifically designed to test sequential memory
(i.e., memory for information units in a specific order) instead of spatial egocentric or allocentric memory.
s0105 Other escape: Barnes maze
p0225 The major disadvantage of water mazes is that they are stressful to animals, significantly increasing corticosterone levels and
distorting findings for experimental manipulations that also increase stress (Harrison et al., 2009). The Barnes maze was designed
to test spatial memory using the negative reinforcement of escape, but without the aversive threat of drowning (McLay et al., 1998).
The apparatus consists of a well-lit circular platform that sports a large number of holes around its periphery. One of the holes is the
opening to an escape tunnel that leads to a home cage; the other holes provide no escape. Both spatial and non-spatial forms of this
task have been developed (Harrison et al., 2006). This task has not yet been used extensively in toxicology, but it is sensitive to the
effects of neonatal methamphetamine exposure (Williams et al., 2003) and modulation of the endocannabinoid system (Harloe
et al., 2008).
s0110 Procedures using positive reinforcement
p0230 Most often, tasks employing positive reinforcement use food or water to reinforce behaviors of interest, although access to
conspecifics (e.g., a nursing dam in the case of an infant, or an estrous female) has also been used. These methods do not require
application of noxious stimuli, but do require depriving the animal of the reinforcing stimulus to generate the motivation to emit
the behavior of interest. Behavior can also be maintained by infusion of some reinforcing drugs (e.g., cocaine: (Roberts et al.,
2007)) and direct stimulation of certain brain regions reinforcing (Wise, 2005).
s0115 Spatial orienting: Radial arm maze (RAM)
p0235 The RAM can be used for a variety of spatial tasks. The prototypical procedure requires animals to distinguish between the locations
of previously visited and unvisited feeding sites during a free-choice test session (Olton and Papas, 1979). The typical RAM consists
of an open, central arena from which several (usually 8) arms radiate like spokes of a wheel. Rats deprived of food are placed in the
central arena and permitted to enter the arms to find food or water located at the end of the arms. In this procedure, the most
effective response strategy is to avoid those arms of the maze from which the food has been removed during a previous entry or in
which food has never been present. Usually, test sessions are terminated after all of the reinforcers have been found or after some
time-limit has been exceeded.
p0240 RAMs can be used with a number of species, accommodate a number of experimental manipulations (for example, delays,
conditioned cues, various patterns of baited and unbaited arms), and assess working and reference memory, or learning. Wirsching
et al. (1984), for example, always put food in the same arms for each daily trial; with repeated trials, food-deprived rats learned to
avoid the arms which never had food. Because the unbaited arms remained constant across the test, solving this problem required
reference memory. This procedure was compared with the original method, in which all arms were baited with food each day and
the animal was required to remember which arms had been visited on that day. Because the critical information in that test changes
from trial to trial, that method assesses working memory. Wirsching et al. found that the anticholinergic muscarinic receptor
antagonist scopolamine selectively disrupted the working memory component of the task without affecting reference memory. The
RAM has been used to study the effects of many compounds on cognitive functioning in rats (Levin, 1988, 2002). Although it has
high ecological validity by relying on rats’ natural foraging strategies, the mazes take up a lot of space and training and testing
animals on RAM tasks can be time-consuming.
s0120 Learning: Discrimination and reversal
p0245 Discrimination learning and discrimination reversal procedures have been used to quantify chemical-induced learning deficits and
deficits in executive functioning, the latter of which depends upon intact functioning of the prefrontal cortex (Welsh et al., 1991).
Reversals can be conducted manually, with animals choosing between physical stimuli—for example, in a sand digging task for rats
and mice (Birrell and Brown, 2000), in operant chambers (Hilson and Strupp, 1997), or in mazes (Ragozzino et al., 1999). In one
version of this task, the experimental animal is presented with two or more stimuli which vary in one or more parameters, for
example, color, position, or pattern, and the animal must respond to one of the stimuli for reinforcement. Once the animal has
learned to respond to the stimulus according to some criterion, the contingency is changed so that responses to the previously
incorrect stimulus are now reinforced.
p0250 One useful feature of the reversal procedure is that a separate learning curve can be generated each time that a reversal is made.
Bushnell and Bowman (1979) reported that young monkeys fed a diet containing lead during the first year after birth showed
deficits on the first of a series of reversals of several tasks involving spatial, color, and form discriminations. Many subsequent
experiments have confirmed that developmental exposure to lead impairs performance of both spatial and non-spatial learning
tasks (Rice, 1993; White et al., 2007). Reversal procedures have also been reported to detect cognitive dysfunction produced by a
number of chemicals from different classes, including metals (Bushnell, 1990; Bushnell and Bowman, 1979; Hilson and Strupp,
1997), pesticides (Raffaele et al., 1990), polychlorinated biphenyls (PCBs) (Schantz et al., 1989) and prenatal cocaine (Garavan
et al., 2000).
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s0125 Sequence learning: Repeated acquisition methods
p0255 Repeated acquisition procedures enable multiple assessments of learning in the same subject. In general, the methods involve
training a subject to solve a kind of problem and then presenting a series of those problems and measuring how quickly they are
learned. Serial reversal learning is an example of repeated acquisition; other forms provide a more stable baseline of acquisition
rates that can be used to assess the effects of drugs and chemicals. One distinct advantage of the method lies in the fact that learning
can be tested repeatedly in the same subject, which enhances statistical power and also permits evaluating the onset and duration of
effects of a chemical, as well as recovery from those effects.
p0260 Bushnell and Angell (1992) used a repeated acquisition procedure in the radial arm maze (originally developed by Peele and
Baron (1988)) to study the effects of TMT on cognitive function of rats. In this experiment, rats were trained to obtain food pellets at
the end of 4 of 8 baited arms of the maze in each of a series of daily 12-trial sessions. The set of baited arms was changed each day,
thus requiring the rats to learn a new set of baited arms in each session. Errors were scored as entries into non-baited arms; errors
decreased across trials within each session as the rats learned which arms were baited and which were not. Rats treated with TMT
showed a significantly slower decline in within-session error rates. Repeated acquisition procedures have also been described for
the Morris water maze (see above).
p0265 Using learning of response sequences in an operant chamber, Cohn et al. (1993) used a multiple schedule containing
components of repeated acquisition and performance to assess cognitive dysfunction in rats following developmental exposure
to lead. In this task, sequences of three responses were reinforced. The correct sequence during the repeated acquisition component
changed with each experimental session. In the performance components, the correct sequence remained constant across sessions,
so new learning was not necessary for accurate responding. Cohn et al. found that developmental exposure to lead significantly
decreased accuracy on the repeated acquisition component, but not on the performance component. These changes were
interpreted to reflect a specific alteration in learning, because normal responding in the performance component meant that the
similar sensory and motor requirements of simple performance of the task were unimpaired. Further analysis of the data indicated
that the effects were associated with an increase in perseverative responding in the lead-exposed rats. This method was also used to
show that learning-specific deficits followed treatment of rats with TMT (Cohn and MacPhail, 1996) and chlorpyrifos (Cohn and
MacPhail, 1997).
s0130 Sequence learning and memory: Three-panel runway
p0270 The three-panel runway was designed by Furuya et al. (1988) to assess working memory in rats. In this device, a wide runway has a
start box at one end and a goal box with a reinforcer (usually food) at the distal end. The runway contains four sequential walls with
three panels each, placed on the left, center, or right third of each wall. Each panel contains a hinged gate that can be locked or left
unlocked for the animal to push open and pass through. In a typical trial, one gate in each wall is unlocked, and the animal must
learn the sequence of open gates that will allow access to the reinforcer at the end of the runway. For example, the sequence of open
gates may be C, L, C, R; attempts to open each gate are recorded. The number of errors (pushing on a locked gate) decreases as the
animal learns the correct sequence across 5 to 10 trials.
p0275 This elegant device has been used frequently since its invention to study mechanisms of working memory in rats, but no studies
designed to assess neurotoxicity have to our knowledge been reported. We describe it here because we see this method as a
promising tool for implementing repeated acquisition tests of learning, for which a modified device has been developed for mice
(Brooks et al., 2000). Indeed, this method could be automated with appropriate sensors and food delivery devices. For example, the
animal could traverse the runway in one direction under learning contingencies (a sequence of gates that changed across test
sessions) and in the other direction under performance contingencies (a constant sequence every test session). This approach
would yield a learning curve for each animal and a measure of sensorimotor capacity from the performance component during
each session.
s0135 Memory
s0140 Working memory: Delayed alternation
p0280 Delayed alternation has been proposed as a method for assessing the development of the septohippocampal cholinergic pathway,
given the critical role of this system in learning and memory (Givens and Olton, 1990). Delayed alternation can be implemented
reliably with rodents in mazes and, with primates, in the Wisconsin General Test Apparatus. For example, Stanton et al. (1994)
conducted delayed alternation tests in a T-maze to assess the cognitive effects of developmental exposure to chlorpyrifos, a long-
acting inhibitor of acetylcholinesterase. In this experiment, rats were trained using a discrete-trial, delayed alternation task. This
involves requiring the food-deprived rat to make a forced choice to enter one arm of the T-maze for food reinforcement. At a later
time, the rats are given a choice trial in which both arms are available for entry, but reinforcement is available only in the arm
alternate to that entered on the preceding forced trial. Rats were also tested on a position discrimination task, in which the rats were
reinforced for consistently selecting one of the two arms. Acquisition of the position discrimination task is not specifically affected
by disruption of hippocampal function. Stanton et al. reported that a single exposure to chlorpyrifos produced a dose- and time-
dependent deficit in delayed alternation, but not in position discrimination, a pattern consistent with deficits in working memory
but not reference memory. These behavioral effects were associated with dose-related inhibition of cholinesterase activity in the
frontal cortex and hippocampus.
p0285 Delayed alternation has also been implemented with rats in an operant lever-pressing environment. In this situation, two
retractable response levers are presented to the animal in a series of trials, and food is delivered if the animal presses the lever that it
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did not press on the previous trial (alternation response). No external cue is provided to indicate the ‘correct’ lever; thus the animal
must base its choice on memory of its previous response. Using this method, Widholm et al. (2004) showed that developmental
exposure of Sprague-Dawley rats to either a mixture of PCBs (Aroclor 1254) or methyl mercury caused a reduction in accuracy that
was not delay-dependent, suggesting that the deficit was not due to memory impairment per se, but could reflect an attentional or
associative impairment. The same group showed that dietary n6 fatty acid deficiency during development also impaired delayed
alternation behavior (Roegge et al., 2005).
p0290 However, this operant alternation method did not result in measurable changes in accuracy across delays in a study of the
developmental effects of inhaled ethanol in Long-Evans rats (Oshiro Q5et al., in press). In this case, the rats appeared to make their
choice for the alternate lever immediately after each response by physically moving to that lever after collecting the food pellet from
the central food well, thus maintaining high accuracy at all delays. The reasons for the discrepancy between this study and those
above are not entirely clear; two possibilities are differences in rat strains and the particular retractable lever used. Neither provides a
satisfactory explanation, and caution is therefore urged regarding use of this task in rats.
s0145 Working memory: Delayed matching
p0295 Delayed matching procedures avoid the problem of positional mediation of the next response in tests of memory. In this
procedure, a sample stimulus (e.g., a tone, color, pattern, or response manipulandum) is presented to the animal and is terminated
after a specified time or by a response by the animal. This is followed by a delay interval, which is ended by the presentation of the
sample along with one or more alternative comparison stimuli. Choosing the stimulus that matches the sample results in delivery
of a positive reinforcer (usually food), while selecting another stimulus usually terminates the trial without the reinforcer and
initiates an intertrial interval. The introduction of a delay between the time when the sample is presented and when it must be
selected from among the set of comparison stimuli makes the delayed matching to sample task a test of short-term memory (Spear
et al., 1990).
p0300 Bushnell et al. (1993) used a delayed matching procedure in rats to assess the effect of acute chlorpyrifos exposure. This
procedure records several measures including accuracy of responses, latency to respond, and responding in the food cup during the
intertrial interval. The method assesses both working memory and reference memory in the same animal during the test session.
Working memory was defined as accuracy on matching trials, and reference memory as accuracy on discrimination trials in which a
cue light indicated the correct response at the end of the delay. Rats received a single injection of chlorpyrifos and were tested for
several weeks thereafter. Chlorpyrifos caused motor slowing as measured by increased response latencies and decreased responses
during the intertrial interval. Matching accuracy was reduced for 2–3 weeks after chlorpyrifos, whereas discrimination accuracy was
not affected, indicating that the pesticide affected working memory specifically. Similar procedures have also been used to study the
cognitive effects of a variety of chemical and environmental manipulations and memory mechanisms in rodents, non-human
primates, and children (Paule et al., 1998).
s0150 Attention
s0155 Five-choice serial reaction time task
p0305 Signal detection methods have been developed to assess sustained and selective attention in humans and experimental animals.
These methods utilize automated testing chambers that can also in some cases be adapted to test constructs mentioned in previous
sections: learning, delayed alternation, discrimination reversal, repeated acquisition, set shifting, and inhibitory control.
p0310 The 5-choice serial reaction time test, or 5-CSRTT (Carli et al., 1983; Robbins, 2002), has been widely used to investigate the
neurobiology of attention, as well as to study the effects of drugs and chemicals on attention and inhibitory control. In this method,
a rat or a mouse is placed in a chamber that has a food cup on one wall and a horizontal array of five illuminable nosepoke ports on
the opposite wall. The animal initiates a trial by opening the food cup door. At some time thereafter, one of the ports is illuminated,
and if the animal “pokes” its nose into that port (i.e., breaks the photobeam crossing the port) within a few seconds, a food pellet is
delivered to the food cup. Analyses of error types (e.g., premature responses, inaccurate responses, errors of omission) provide
insight into whether sustained or selective attention or inhibitory control are affected, and measures of response latency and latency
to collect rewards provide indices of motivation and motor function.
p0315 The neurobiological and neurochemical bases of performance in variations of the 5-CSRTT task have been explored using
lesions and pharmacological manipulations (Dalley et al., 2002, 2004; Robbins, 2002; Robbins and Roberts, 2007). In addition, a
version of this task was used to uncover deficits in the rapid engagement of attention as a result of prenatal cocaine exposure
(Morgan et al., 2002); impairments in sustained attention in animals exposed chronically from birth to the flame retardant DE-71,
a commercial mixture of polybrominated diphenyl ethers (Driscoll et al., 2009); and impairments in inhibitory control in animals
exposed during lactation to lead (Stangle et al., 2007).
s0160 Two-lever signal detection task
p0320 An alternative signal detection method, using more traditional operant equipment, was developed to study sustained attention in
rats (Bushnell et al., 1994). In this test, two retractable levers that flank a central food cup are periodically inserted into an operant
chamber. A press on one lever produces food if a centrally-located signal light has flashed briefly since the previous trial, and a press
on the other lever produces food if no signal has occurred. Both levers retract after a single press has been made. The occurrence of
the signal is temporally unpredictable, which requires the rat to attend to its occurrence to respond accurately.
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p0325 Because a lever press is required in every trial, success is determined by which lever is pressed rather than by whether a lever is
pressed. For this reason, a response failure does not occur because the animal was not attending to the signal, but instead reflects
reduced motivation or motor capacity. This task thus yields less ambiguous evidence for attention deficits than does the 5-CSRTT,
in which an omission error could be due to attentional, motivational, or motor deficits. The 2-lever method has been used to assess
effects of inhaled organic solvents (Bushnell, 1997; Bushnell et al., 2007; Oshiro et al., 2001), pesticides (Bushnell et al., 2001;
Samsam et al., 2005); PCBs (Bushnell et al., 2002; Geller et al., 2001), and algal toxins (Rezvani et al., 2001). In addition, a variety
of drug and lesion studies have yielded a substantial neurobiological database of the processes controlling behavior in the task
(Sarter et al., 2005), and application of a neural network model has illustrated the cognitive processes involved in its performance
(Schmajuk and Bushnell, 2009).
s0165 Executive function
s0170 Set-shifting methods
p0330 A conceptual extension of the reversal learning procedure involves requiring the subject to shift responses across stimulus
dimensions or sets, and engages higher-level executive functions to execute accurately. These tests are modeled after the Wisconsin
Card Sorting test, which was designed to assess cognitive flexibility, or set-shifting, in humans (Dehaene and Changeux, 1991;
Milner, 1963; Parks et al., 1992). In set-shifting methods, subjects are presented stimuli that contain exemplars of more than one
dimension (e.g., size, color, and shape). Thus, in a two-choice discrimination trial, a particular pair of stimuli might be a large, blue
square and a small, red circle. In this trial, color (e.g., red) might be the correct attribute during the initial discrimination, with the
other dimensions (size and shape) being irrelevant. After the subject has learned this discrimination, the contingency for
reinforcement could be changed in an intra-dimensional shift (now blue is correct, staying with the color dimension) or in an
extra-dimensional shift (e.g., now square is correct, shifting to the shape dimension). The number of trials or errors that are
required for the subject to reach a criterion of accuracy on each shift or reversal provides a measure of the animal’s cognitive
flexibility. This method has been used to study the effects of CNS lesions with a sand-digging method (Birrell and Brown, 2000;
Dias et al., 1996; McAlonan and Brown, 2003) and prenatal cocaine with a 3-choice olfactory discrimination method (Garavan
et al., 2000). However, the effects of toxic chemicals on performance in these tasks in animals have not been explored to date.
s0175 Go no-go and stop signal tasks
p0335 Cognitive control is an aspect of executive functioning that includes processes such as action selection and initiation (particularly in
reward-based learning), inhibition of responses, delay of gratification, and monitoring of performance (Ridderinkhof et al., 2004).
Although these processes are neuroanatomically and neurochemically separable (Eagle et al., 2008), they have traditionally been
explored as a singular phenomenon in both human and non-human animals using go no-go and stop signal tasks.
p0340 Both tasks consist of many trials, each of which begins with the presentation of a stimulus (a “go” signal) to which the subject
has been trained to make a response for a reward. However, on a subset of trials, the subject also receives a signal to inhibit
responding (a “no-go” or “stop” signal), and the response must be withheld in order for the subject to receive the reward. Two
features differ between the go no-go and stop signal tasks: the timing of the presentation of the “no-go” or “stop” signal, and the
presence or absence of a decision-making component. In the go no-go task, the subject receives the “no-go” signal before or during
the “go” signal, thus giving the subject time to process a cognitive decision regarding whether or not to respond. The stop-signal
task differs in that the “stop” signal is presented after the stimulus, and sometimes even during the animal’s execution of the “go”
response. In fact, the timing of the “stop” signal is manipulated to measure the subject’s speed of physically inhibiting its ongoing
response (also referred to as “action inhibition”). Under these conditions, advance decision-making (“action restraint”) regarding
whether the response should be executed or withheld is not possible.
p0345 Performance in both the go no-go and stop signal tasks is impaired in individuals with attention deficit hyperactivity disorder
(ADHD) (Rubia et al., 2007) and in chronic stimulant abusers (Fillmore and Rush, 2002). Unfortunately, these tasks have not been
heavily utilized in behavioral toxicology, but evidence of inhibitory control deficits in humans (Stewart et al., 2005) and rats
(Widholm et al., 2001) exposed to PCBs, as shown in other tasks, suggests that these tasks would be sensitive to the effects of these
and other chemicals.
s0180 Issues in the Interpretation of Results
s0185 Nonassociative vs. Associative Factors
p0350 In behavioral toxicology, chemical-induced alterations in cognitive functions must be inferred from changes in behavior. There-
fore, it is necessary to understand the factors that control behavior, whether cognitive or not. Both respondent and operant
conditioning methods depend upon the relationship between stimuli in the environment and specific conditioned behaviors. If a
chemical alters the animal’s perception of those stimuli, or its ability to perform the required motor response, then behavior may be
altered because of these nonassociative factors. Respondent behaviors are particularly susceptible to the magnitude and duration of
the eliciting stimulus, and chemicals that diminish its detection will influence the outcome measure (e.g., the frequency of a CR),
whether or not cognitive impairment is also present.
p0355 Conditioned behavior is also strongly influenced by antecedent conditions, such as the past history of reinforcement and the
current motivational state of the animal. In addition, other variables, such as the type of reinforcer, the magnitude and duration of
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reinforcement, and programmed contingency between response and reinforcer, affect the probability or accuracy of responses.
Chemically-induced alterations in motivation and sensory and motor abilities will also affect measures of conditioned behavior,
regardless of possible effects on cognitive function. Other examples of nonassociative factors include state-dependent effects,
interactions with stress, and alterations in exploration or response bias. Finally, effects of chemicals on cognitive function are
sometimes task-specific: that is, a deficit in learning may be observed using one type of task, but may not be detected using a
different task.
p0360 In addition to non-associative factors influencing the interpretation of a behavioral change in a test designed to assess a
cognitive function, caution should be exercised in imputing a deficit in a specific cognitive process to a toxicant-related change in
behavior. As noted above, learning, memory and attention do not operate independently, so even if the study provides reasonable
assurance that a robust change in behavior occurred and that sensory and motor effects were not responsible for it, the culpable
cognitive process may still be debatable. Examples of these interpretive issues are given below.
s0190 Examples of Problems of Interpretation
s0195 Altered sensory function
p0365 Environmental stimuli, such as lights or tones, are frequently used to control or manipulate behavior. Extra-maze cues are essential
for performing spatial tasks in the Morris water maze and radial-arm maze. Noxious stimuli, such as electric shock, are applied to
the grids of the test chamber to motivate escape behavior. Chemicals that affect perception of these stimuli could clearly affect the
outcome of studies on learning and memory without affecting cognitive function directly. In a study by Lee and Rabe (1992), it was
observed that rats exposed to MAM during gestation showed a marked deficit in learning to locate a hidden platform in a Morris
water maze. One interpretation of these results is that exposure to MAM during development reduced the rat’s cognitive capacity to
acquire and use spatial information. Lee and Rabe, however, point out that, in addition to its effects on development of the brain,
MAM also damages the visual system in rats (Ashwell, 1987), which impairs their ability to discriminate visual patterns. Therefore,
because learning and performance in the Morris water maze depends on use of visual extra-maze cues, it is likely that the inability to
use appropriate visual cues to solve the maze contributed to the observed behavioral deficits.
p0370 Other tests have been designed to provide independent evidence for changes in sensory, motor, and cognitive functions. These
methods provide a way to determine whether the observed effects of a treatment arise from changes in cognitive function or not.
For example, in the signal-detection method developed by Bushnell et al. (1994) for assessing sustained attention, the signal
intensity is systematically varied during each session. This manipulation yields a psychophysical function that relates accuracy of
reporting the signal [P(hit)] as a function of its intensity, which provides evidence for the animal’s sensitivity to the light. Thus, a
horizontal shift in this function indicates a change in threshold for detecting the signal (sensory change), whereas a vertical shift
indicates a change in attentiveness to the signal. An increase in the false alarm rate also provides evidence for attentional
dysfunction, particularly if it accompanies a downward shift in the function relating P(hit) to signal intensity. Acute exposure to
many drugs (Bushnell et al., 1997) and organic solvents (Bushnell, 1997; Bushnell et al., 2007; Oshiro et al., 2001) cause vertical
shifts in these parameters (suggesting attention deficits), whereas horizontal shifts suggesting sensory threshold shifts were
observed after exposure to chlorpyrifos (Bushnell et al., 2001) and the PCB mixture Aroclor 1254 (Geller et al., 2001).
s0200 Altered motor function
p0375 In order to assess cognitive function using behavioral tests, the ability of the animal to perform the designated response must be
taken into consideration. For example, Llorens et al. (1993a,b) tested the effects of IDPN on acquisition and performance of rats in
several behavioral tests. The retention of passive avoidance and effects on the acquisition of a food-reinforced response in the RAM,
steady-state performance in the RAM and RAM repeated acquisition were significantly affected by IDPN. It was difficult to assess
learning capabilities in the Morris water maze, however, because the there was a dose-related impairment in swimming ability.
It was hypothesized that vestibular toxicity produced by IDPN affected their ability to swim, precluding the assessment of cognitive
function using this test.
p0380 More subtle changes in motor capacity can also be (mis)-interpreted as changes in cognitive function. For example, Bushnell
(1988) observed that rats that were trained to press a retractable response lever for food reward in an autoshaping procedure
acquired the response more quickly when tested immediately after an acute exposure to p-xylene than did control rats. This
apparent facilitation of learning was examined further. Because the intoxicated rats were motorically more active than controls in
photoactometers and were observed to be physically unsteady after exposure to the solvent, the possibility that the enhanced
learning was caused by uncoordinated activity was tested. In support of this hypothesis, increasing the force necessary to press the
lever eliminated the facilitative effect of exposure, suggesting that the treated animals did not learn more quickly than controls, but
simply encountered the contingency between pressing the lever and obtaining food sooner, by virtue of inadvertent lever-pressing
due to clumsiness. Further, use of an automaintained reversal learning procedure permitted independent measures of activity (rate
of lever pressing) and learning (accuracy of pressing). Inhalation of p-xylene reduced overall lever-pressing rates but did not affect
the rate of acquisition of the correct response, confirming that the effects of the solvent on behavior were motoric and not cognitive.
s0205 Task specificity
p0385 As discussed previously, there are several types of memory, and if each has its own neurobiological substrate, then neurotoxicants
might affect some measures of memory but not others. An example of this can be found in a study of the effects of repeated
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exposure to the cholinesterase inhibitor disulfoton (Llorens et al., 1993a,b). In these experiments, rats were injected for 30 days
with various doses of disulfoton and tested for cognitive dysfunction in the Morris water maze and passive avoidance procedures.
Disulfoton affected the acquisition of the spatial memory task, but had no effect on acquisition or retention of passive avoidance.
p0390 Organophosphate pesticides appear to impair working memory but to spare reference memory. For example, Bushnell
et al. (1991) developed a delayed matching-to-position test for rats that included both matching trials, in which the rat was
required to remember the location of the sample stimulus after a delay, and discrimination trials, in which the correct response was
cued by a light after the delay. Both diisopropylfluorophosphate (Bushnell et al., 1991) and the pesticide chlorpyrifos (Bushnell
et al., 1993) reduced choice accuracy on matching trials, but not on discrimination trials. These results indicate that the accuracy
deficits were unlikely to be associated with memory for the response rule, or for information that remains constant during the
assessment, i.e., information for which reference memory is engaged. In contrast, working memory, i.e., memory for information
that changes frequently (in this case, the identity of the sample lever), was impaired by exposure to the chemicals.
p0395 As described above, Cohn et al. (1993) used a similar strategy of combining multiple tasks into an assessment tool for learning
impairment. In their method, repeated-acquisition and performance components alternated in each test session. This method yields
semi-independent measures of learning and sensorimotor function, which was used to argue that effects of drugs, lead and chlorpyrifos
are specific for learning. However, it may also be argued that the increased sensitivity of the learning component in this method, and of
the matching component of the combined delayed matching and discrimination tasks in Bushnell et al. (1991) procedure, can be
attributed to the greater difficulty of those components per se, and not to the specific CNS process required to perform the task.
s0210 Cognitive Science and Toxicity Screening
p0400 Concern about the potential risk to public health of the large number of untested anthropogenic chemicals in the environment has
spurred development of high-throughput molecular and cell-based tests designed to screen these chemicals for toxicity (Gibb,
2008; NRC, 2007). Given that this approach cannot accommodate complex behavioral tests in whole animals, opposing concerns
have arisen regarding the sufficiency of this strategy to protect public health. These concerns have prompted discussions about the
necessity of these tests and their role in the assessment of risk of chemical exposure (Bushnell, 2014; Bushnell et al., 2010; Driscoll
and Strupp, 2015). Whereas opinions vary regarding the importance of these tests, it is clear that the new high-throughput strategy
is not yet developed sufficiently to replace assessments of behavior in whole animals at this time. Whereas simpler whole-animal
screens are often sufficient for detecting chemicals that cause cognitive dysfunction (Bushnell, 2014), a great deal of work will be
needed to develop high-throughput methods that can adequately assess effects of chemicals on the complex, interactive processes
that underlie cognitive functions in living animals.
p0405 Bushnell et al. (2010) outlined some challenges and opportunities for behavioral scientists in this difficult and important
endeavor. Challenges include predicting behavior using computer models of complex neurobiological pathways; standardizing
study designs and dependent variables to facilitate creation of databases for use in meta-analysis; and decreasing the cost and
increasing the efficiency of behavioral assessments. Opportunities include identifying and characterizing toxicity mechanisms and
pathways impacting cognitive function; informing the conditions and limits of extrapolation across species, time, and dose;
addressing issues of susceptibility and variability; providing reality-checks on selected positives and negatives from screens; and
performing targeted testing and dose-response assessments of chemicals flagged during screening. Without advances in these areas,
even the most thorough screening process will not yield information that can protect the sophisticated cognitive functions that
enable the many enterprises and endeavors of human culture.
s0215 Summary and Conclusions
p0410 Humans exposed to chemicals demonstrate alterations in cognitive function, including confusion, disorientation, and deficits in
attention, learning and memory. Conceptually, it can be difficult to separate these cognitive processes, although they can be
operationally defined by the procedures used to assess them. A number of procedures have been used by neurotoxicologists to
detect and quantify the cognitive effects of chemicals in animal models; these procedures can be classified generally as being
designed to assess nonassociative or associative processes. Examples of tests of nonassociative learning include habituation and
sensitization, whereas examples of tests of associative learning include methods that quantify conditioned behavior using positive
or negative reinforcement in operant or maze environments. Examples of tests that use negative reinforcement include shock
avoidance or escape from water. Procedures commonly used in neurotoxicology that employ positive reinforcement include tests of
food retrieval in the radial arm maze, autoshaping, discrimination and reversal learning, alternation tasks, delayed-matching-
to-sample, and signal detection. Because of the wide variety of processes necessary for attention, learning and memory, no single
task may be sufficient to assess chemically-induced cognitive dysfunction by itself. More than one test may be necessary to
determine whether a chemical does or does not affect cognition and to characterize the cognitive process(es) that are affected.
Finally, the interpretation of results from tests of cognitive function must be considered in the context of other potential changes in
nervous system function: that is, changes in sensory capabilities, motor function, motivation, and response strategy. Neurotox-
icological research in this area is closely linked to a better understanding of the neurobiology of learning, memory and attention.
Future investigations should attempt to use the available knowledge about the putative sites of neurotoxic effects and underlying
neurobiological bases of the various forms of cognitive function.
Cognitive Function 13
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