Cognitive Function☆

PJ Bushnell, U.S. Environmental Protection Agency, Durham, NC, USA

LL Driscoll, Colorado College, Colorado Springs, CO, USA
ã 2015 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
Flavor aversion 5
Eye blink 6
Trace fear 6
Instrumentally Conditioned 6
Procedures using negative reinforcement 6
Procedures using positive reinforcement 8
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

Introduction

The fact 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, mathematical abilities, categorization, coding, concept shifting, distractibility,
memory, pattern recognition, reading, spatial relations, sustained and selective attention, vocabulary, 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)).
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).

☆
Change History: P. Bushnell and L. Driscoll rewrote every section of the chapter to bring the whole work up to date. They included new sections on habituation
and sensitization, trace fear conditioning, and executive function, and greatly expanded the discussion of water maze methods. They removed Tables 1 and 3
and rewrote and reformatted the previous Table 2 to the curent Table 1 and added a ‘Summary and Conclusions’. They added a section on ‘Cognitive Science
and Toxicity Screening’ (a) to provide cautions about interpretation and application of data generated by new high-throughput toxicity test methods, and (b) to
identify some challenges and opportunities for behavioral scientists in this new testing environment.

Reference Module in Biomedical Research http://dx.doi.org/10.1016/B978-0-12-801238-3.02206-6 1

2 Cognitive Function

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.
The purpose of this chapter is to facilitate these efforts in several ways. 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 chapter 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.
Due to space limitations, this chapter 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, 2011b) and C. elegans
(Bessa et al., 2013; Rankin et al., 1990; Sasakura and Mori, 2013).

Definitions
Principles of Behavioral Analysis
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 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.
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.
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.
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 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. The process of changing behavior by application of reinforcement
contingencies is referred to as operant or instrumental conditioning.

Cognitive Functions: Learning, Memory, Attention and Executive Functions

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,
 Cognitive Function 3

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.
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 (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 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.
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).
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. Executive
functions are also dissociable in rat prefrontal cortex, although the circuitry differs between humans and rats (Uylings et al., 2003).
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.
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 cognitive 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).
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.

Methods for Assessing Chemical-Induced Cognitive Dysfunction

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
apparatus 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.
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
4 Cognitive Function

Table 1 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
Observation
Sensitization Kindled behavioral responses
Associative learning
Classical Flavor aversion Water-bottle test
Eye-blink Conditioning chamber
Trace Fear Conditioning chambers
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

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.

Nonassociative Learning
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).

Habituation
Habituation reflects a reduction in exploration of 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 1 of a flame retardant, polybrominated 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).
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.
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
 Cognitive Function 5

technique have been described for 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).
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 1/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 2 identical plastic objects,
for 5 min. Then, after a 1-hr 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 that
atrazine caused a deficit in recognition memory.

Sensitization
Behaviorally, sensitization manifests itself as the opposite of habituation: an initial response to a stimulus becomes more
exaggerated, rather than attenuated, with repeated exposure to the stimulus. 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.
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 1–2 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, naı̈ve 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.

Associative Learning
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 of 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.

Classically Conditioned
Flavor aversion
An example of a classical conditioning paradigm used to assess memory is the flavor aversion procedure, which is based on the
finding that animals will avoid consuming solutions with flavors previously paired with illness. Peele et al. (1989) exposed rats to
trimethyltin (TMT) and assessed them 30 d 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. Whereas research continues to understand the biological basis of flavor aversion conditioning
(Miranda, 2012; Sandner, 2004), this method of assessing the effects of chemicals on memory has not to our knowledge been
pursued.
6 Cognitive Function

Eye blink
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.
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).

Trace fear
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, whose activity 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.
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.
Results showed that PTU treatment impaired both context and cue learning, but only when the distractor stimuli were presented
during training. Without the distractors, the two low doses of PTU increased freezing behavior (suggesting better learning), and the
high-dose group did not differ from controls. 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.
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.

Instrumentally Conditioned
Procedures using negative reinforcement
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.

Passive avoidance
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 d 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
 Cognitive Function 7

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.
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 organophosphorous 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).

Active avoidance
In contrast to passive avoidance tasks in which withholding a response is reinforced, active avoidance tasks require that the animal
performs 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.
Active avoidance tests have been used recently to assess the effects of diet on the toxicity of the nerve agent soman in adult rats
(Myers and Langston, 2011), early postnatal exposure to diesel exhaust particles (Yokota et al., 2011), and 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.

Water escape: Morris water maze

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 the animal’s view) platform in order to escape
the water; therefore, escape is the negative reinforcer. Swim path length, swim speed and heading direction are measures obtained
either by human observers or video tracking methods to assess the animal’s performance. Early studies (Morris, 1984) showed that
the rats used visual cues outside the maze – typically the walls and configuration of the room that houses the maze – to navigate to
the platform.
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 depending on whether the
animal is allowed to utilize extra-maze (allocentric) cues or must rely on positional (egocentric) responding to determine the
position of the submerged platform in the maze (McDonald and White, 1993). Other variants of the task can be used to
differentially assess functioning of the hippocampal, striatal, 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).
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).
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 (Llorens et al., 1993a), toluene (von
Euler et al., 1993), and lead (Nihei et al., 2000).

Water escape: Biel and Cincinnati mazes

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
8 Cognitive Function

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
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.

Other escape: Barnes maze

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).

Procedures using positive reinforcement

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 drugs (e.g., cocaine: (Roberts et al., 2007)) and direct
stimulation of certain brain regions is also positively reinforcing (Wise, 2005).

Spatial learning and memory: Radial-Arm Maze

The Radial-Arm Maze (RAM) is a device that can be used to present 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.
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.

Learning: Discrimination reversal

Discrimination learning and discrimination reversal procedures have been used to quantify chemical-induced learning deficits and
deficits in executive functioning. 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.
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).
 Cognitive Function 9

Sequence learning: Repeated acquisition methods

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.
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 trimethyltin 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
trimethyltin showed a significantly slower decline in within-session error rates. Repeated acquisition procedures have also been
described for the Morris water maze (see above).
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 trimethyltin (Cohn and MacPhail, 1996) and chlorpyrifos
(Cohn and MacPhail, 1997).

Sequence learning and memory: Three-Panel runway

The three-panel runway was designed by Furuya et al. (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.
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.

Working memory: Delayed alternation

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.
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
10 Cognitive Function

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).
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 et al., 2014). 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.

Working Memory: Delayed Matching

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 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).
Bushnell et al. (1993) used a delayed matching procedure in rats to assess the effects of the pesticide chlorpyrifos on working
memory. This procedure records several measures including correct responses, latency to respond, accuracy, 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 and not reference
memory. 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).

Attention: Five-choice serial reaction time task

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 evaluate constructs mentioned in
previous sections: learning, delayed alternation, discrimination reversal, repeated acquisition, set shifting, and inhibitory control.
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’ into that port (i.e., breaks the photobeam crossing the port) within a few seconds, a food pellet is delivered
to the food cup. Retrieval of the food pellet by the animal initiates the next trial. 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 the food pellets provide indices of motivation and motor function.
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., 2004; Dalley et al., 2002; 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).

Attention: Two-lever signal detection task

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.
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,
 Cognitive Function 11

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).

Executive function: Set-shifting methods

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 perform 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.

Executive function: Go No-Go and stop signal tasks

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.
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.
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.

Issues in the Interpretation of Results

Nonassociative vs. Associative Factors
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 a 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.
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
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
12 Cognitive Function

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.
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.

Examples of Problems of Interpretation

Altered sensory function
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,
since 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.
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).

Altered motor function

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., 1993b) 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.
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 vapor 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.

Task specificity
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
exposure to the cholinesterase inhibitor disulfoton (Llorens et al., 1993a). 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.
 Cognitive Function 13

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.
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.,
1991,1993) 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.

Cognitive Science and Toxicity Screening

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). 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.
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.

Summary and Conclusions

Humans exposed to chemicals report 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, while 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, motor, motivation, and response strategy. Neurotoxicological 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.
14 Cognitive Function

This manuscript has been reviewed by the National Health. Environmental Effects Research Laboratory U.S. Environmental
Protection Agency approved for publication. Approval does not indicate that the contents reflect the views of the Agency nor does
mention of trade names or commercial products constitute endorsement or recommendation for use. We thank Edward D. Levin
and Wendy M. Oshiro for reviews of a draft of this article.

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