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Levin ED, Buccafusco JJ, editors. Animal Models of Cognitive Impairment. Boca Raton (FL): CRC Press/Taylor & Francis; 2006.

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Animal Models of Cognitive Impairment.

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Chapter 12Assessments of Cognitive Deficits in Mutant Mice

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Although most behavioral experiments have been conducted in rats, mice are rapidly becoming the preferred rodent of study in many labs because their genetics are well known, their genome has been sequenced, and they can be genetically manipulated. To date, several different approaches have been used to generate a behavioral phenotype for study.

In “forward” genetics, the analysis proceeds from phenotype to genotype. This is a classical approach, and it includes mice where spontaneous mutations have been identified in certain genes [1] or where mice have been subjected to radiation [2] or chemical mutagenesis [3]. This approach also pertains to mouse strains that have been shown to display a certain phenotype or to animals that have been selectively bred for a given behavioral trait [4–6]. Although forward genetics can provide very interesting animal models, the genetic basis of the abnormal behavior is often obscure. This limitation requires the site(s) of the mutation(s) to be mapped and sequenced [7–9], which can be quite laborious and time consuming. Consequently, many investigators have adopted “reverse” genetics, where the analysis proceeds from genotype to phenotype. Here, a specific gene is targeted for disruption or modification, and the mutants are evaluated for behavioral abnormalities. Investigators typically employ transgenesis to produce either gain of function through expression of hybrid genes and duplication of endogenous genes or loss of function by expressing dominant-negative hybrid genes, toxic genes, or disrupting endogenous genes [10–14]. These gene-targeting approaches in embryonic stem cells, or in one-cell embryos, may lead to alterations in expression of other members of the same gene family, with behavioral compensation occurring during development and adulthood [15]. This developmental compensation is a common criticism of transgenic experiments. However, it should be emphasized that such compensation is rarely the basis of study in mutant mice per se, and there are many incidences where compensation by nonmutant family members does not appear to contribute to the phenotype [16]. Nevertheless, to obviate this criticism of developmental compensation, some investigators have begun using systems that induce or suppress expression of specific genes at certain ages or within a given brain region [17–19]. More recently, reduction in gene expression in vivo has been accomplished through the introduction of RNA interference that targets a specific RNA species [20]. Although this approach does not completely suppress expression of the target gene, it can reduce it (80%) to levels sufficient to produce quantifiable biochemical and behavioral changes.

Together, forward and reverse genetic approaches have provided important insights into the roles that selected genes play in the composition of a given behavioral phenotype. Most of these approaches have some limitations because behaviors in humans are controlled not by a single gene, but by many genes interacting in concert with the environment. Analyses are currently proceeding where (a) qualitative trait loci in mice are identified, (b) mice with known genetic mutations are outcrossed to other mutants, or (c) mice with known genetic backgrounds are exposed to differing environmental conditions [21–23]. This multitude of approaches with mice has and will continue to yield novel insights into the genetic and molecular antecedents that affect behavior.

Preliminary Phenotypic Screening

Behavioral assessment in mice has come a long way in the past century with increasing refinement of paradigms [24–27]. The behavioral phenotype of mice is complex; consequently, these animals should first be evaluated on numerous behavioral domains that include the animal’s general health and well-being, reflexive and motor capabilities, emotionality, anxiety, affective and social behaviors, consummatory responses, and learning and memory. Partially due to the use of mutant mice, the past decade has witnessed numerous advances in identification of molecular mechanisms that underlie a wide variety of behaviors in animals. Over the years, learning and memory processes have received the greatest emphasis for study. Investigators have produced many strains of mutants to examine the roles of different genes in cognitive behavior. However, the focus on this domain of behavior neglects the other possible responses for these animals, some of which may influence performance on cognitive tasks. For instance, N-methyl-D-aspartate (NMDA) receptors have been shown to play an important role in hippocampal long-term potentiation [28], and administration of a NMDA receptor antagonist into the lateral cerebral ventricles impairs spatial memory [29]. Indeed, disruption of the NR1 subunit gene of the NMDA receptor is lethal [30, 31]. On the other hand, mice with targeted disruption of this gene in the CA1 region of the hippocampus survive but show severe impairment in spatial learning and memory [32]. Knockdown of the NR1 subunit also produces a schizophrenia-like phenotype [12]. Hence, it is important that investigators explore the behavioral phenotypes of their mice more completely in the course of cognitive testing, as additional behavioral deficiencies may contribute to the impairments in learning and memory.

To deal with this concern, we have designed a broad series of tests that are administered to all mice brought for testing into the Mouse Behavioral and Neuroendocrine Analysis Core Facility at the Duke University Medical Center. These assessments include basic tests of sensory and motor function, neurophysiological status, and emotionality. These tests can be conducted in the week before beginning cognitive testing (Table 12.1). In our experience, we have found this test battery to be informative because results from these tests often provide clues as to the proper control experiments that should be run for subsequent investigations [13, 33, 34]. Additionally, results from this initial behavioral screen often allow us to select additional behavioral domains for further study. For the purposes of the present chapter, cognitive behavior will refer to the ability of the mouse to acquire, process, store, retrieve, and act upon information gathered from the environment. The techniques and observational methods necessary to investigate cognitive function in mice will be examined and discussed. Given that the history of cognitive testing in the mouse encompasses eight decades of research, it is far too ambitious to describe all of the cognitive tests presently used for mice. Instead, we will concentrate upon tests that we have found to be useful in the Mouse Behavioral and Neuroendocrine Analysis Core Facility at Duke University Medical Center.

TABLE 12.1

TABLE 12.1

Preliminary Behavioral Screening Prior to Cognitive Assessment

Anxiety

The first test that we typically administer to mice first entering the Core Facility is the zero maze. The elevated zero maze was initially described and pharmacologically validated for anxiety-like behaviors in mice by Shepherd and colleagues [35]. The maze is elevated approximately 18 in above the floor and consists of a circular runway divided into two open and two closed quadrants (Figure 12.1). A camera is mounted directly over the apparatus to allow video-taping of behavior for later analyses with programs such as the Noldus Observer or Ethovision (Noldus, The Netherlands). Lighting for the maze should be indirect and even, but between 40 to 60 lux when a light meter is placed level to the surface of the open arm. The apparatus and mouse are shielded from the observer and any other activity in the room by a wall or curtain. In our testing facility, we have found that the best and most reliable results are obtained when the animals are brought into the room several hours before testing and the room remains undisturbed until completion of testing. The mouse is transported to the maze in a solid-bottom container (pipette box) and is placed into the closed arm of the maze facing the wall. The animal is allowed 5 min to freely explore the apparatus. Behavioral measures include the amount of time spent in the open areas, number of entries into the open areas, and the frequency of traversing from the closed area through the open area to the opposite closed area. In addition, the frequency of head dipping over the edge of an open area, rearing, and stretch-attend postures (rear feet in closed arm but body stretches forward such that the front paws are in the open area) are scored, and these ethological behaviors signify measures of “risk-assessment” or exploration by the animal. We also score the time the animal spends freezing and grooming in the apparatus. Animals with anxietylike phenotypes typically exhibit a low propensity to explore the open areas relative to control animals, and may also demonstrate reduced frequencies of the risk assessment or exploratory behaviors with or without enhanced freezing or grooming [36, 37].

FIGURE 12.1. Zero maze for testing anxietylike behaviors.

FIGURE 12.1

Zero maze for testing anxietylike behaviors. The zero maze is elevated approximately 18 in above the floor, and it consists of two open and two closed areas. Mice are permitted free exploration of the maze for 5 min. Illumination of the maze is critical (more...)

In the event a given mouse genotype presents an anxietylike phenotype, responses to anxiolytics are examined, and the animal is evaluated in other tests of emotional responses that include assessments of “depressivelike” behaviors, social interaction, drug abuse, and fear conditioning.

Open Field

Several days following zero-maze testing, spontaneous activities of the mice are examined in the open field for 1 hour. Many types of open fields are commercially available for mice, and they typically involve a large arena that can be divided into smaller areas. The lighting across the floor of the arena should be even, and we typically test our mice at 350 to 600 lux [37]. Both horizontal (locomotion) and vertical activities (rearing) are monitored by infrared beams, and this information is relayed to a computer with software that automatically records the location and activity of the animal. Behavioral output includes the numbers of horizontal beam-breaks or the distance traveled and the number of vertical beam-breaks or rears. In addition, activities in the center and peripheral zones of the open field as well as activity maps can be generated. Open-field testing typically begins by placing the animal into one of the four corners of the arena and allowing the mouse to freely explore the arena. Overall reduced activity in the open field may be indicative of motor impairment or weakness, particularly if little or no rearing behavior is observed. Alternatively, low activity can also indicate increased anxiety or neophobia. These latter behaviors may be associated with reduced time spent in the center of the open field and increased time spent in the corners or along the perimeter of the arena. Analyses of the behaviors in the open field can be quite informative in planning subsequent testing, particularly when motor disturbances or ataxia may be present [16, 38, 39] or when monoaminergic function may be perturbed [13, 37].

Neurophysiological Screen

The neurophysiological screen for mice is a series of very short tests used to assess several dimensions of neurological functioning and behavior that include sensory and motor function, autonomic reflexes, emotional responses, and rudimentary cognition [13, 16, 27, 34, 40]. Although procedures may differ among laboratories, most tests are based upon the original behavioral screen described by Irwin [41]. The tests are conducted in five phases that consist of 36 discrete measures (Table 12.2) scored on a multiple point scale (Table 12.3). In addition to the tests from the neurophysiological screen, our laboratory also evaluates grip strength by automated meter. Although our neurophysiological screen is a comprehensive test battery, it is not exhaustive, and additional tests can be incorporated into the screen (depending upon the mutation) or be administered later. For example, Abi2 homozygous mutants (knockout [KO] mice) have eye defects [33]. Since many tests for cognitive performance in mice rely upon vision, it was important to conduct a detailed neurophysiological examination of vision to determine whether the KO mice respond to light, have depth perception, and can discriminate patterned stimuli. Pupillary responses to light were first evaluated. The mouse was held by hand and a 1.13-W pen light was shined into the eye for 3 sec, followed by a 5-sec intertrial interval over three trials. The mice were filmed with a high-resolution video camera, and the film segments were digitized (Dazzle Video Creator, Pinnacle Solutions, Mountain View, CA) and analyzed frame by frame for each animal’s response. No differences between wild type (WT) and KO mice were observed. We next tested responses to light and dark by placing the mice in a passive-avoidance apparatus. No genotype differences were noted in the latency for the mice to leave a lighted compartment and enter the darkened chamber, or in the time spent in each of the chambers. These data suggested that both genotypes could discriminate light from dark and that levels of emotionality were similar between the animals. To examine depth perception, we slowly lowered the mice to the bench top. Both WT and KO mice extended their forepaws upon being lowered to the lab bench. Finally, we tested whether the mice could track a moving object within the visual field. As no genotype differences were discerned in any of the tests for vision, we concluded that rudimentary vision was not impaired in these KO mice.

TABLE 12.2

TABLE 12.2

Phases and Tests of Neurophysiological Screen

TABLE 12.3

TABLE 12.3

Scoring for Neurophysiological Screen

Cognitive Testing

Task selection is perhaps the most crucial decision an investigator can make. However, with the multitude of cognitive paradigms available for mice and the availability of mutant animals, it becomes important for the experimenter to decide a priori which tests are the most suitable for study. As the behavioral phenotype of many mutants may be heterogeneous, it is critical to examine multiple aspects of cognition that cover different domains of functioning, including preattention and attention, and various aspects of learning and memory (Figure 12.2). Tests of preattentive functioning have been described for mice [42, 43], and most utilize a simple testing paradigm called prepulse inhibition (PPI). Additional paradigms include simple screens using object discrimination tests [37, 44] or more complex paradigms such as go/no-go testing [45–46], five-choice serial attention tasks [47], or latent inhibition [48, 49]. Finally, tests of learning and memory can be designed to assess more specific areas of functioning, including associative learning, nonspatial or spatial learning, short- and long-term memory, as well as neurologically specific deficits as revealed by fear or eyelid conditioning. As testing across multiple cognitive domains is preferable and because the numbers of mice available for testing may be limited, the investigator may need to adopt two different strategies. First, he/she should be mindful of the order in which the behavioral tests are administered, especially if they are given in series. Under a multiple-test regimen, the least stressful tests are conducted first. Second, once behavioral deficits have been identified, it is important to replicate these results in naïve mice so that prior test experience can be excluded as a confounding variable.

FIGURE 12.2. Cognitive testing for the mouse.

FIGURE 12.2

Cognitive testing for the mouse. There are many cognitive paradigms available for assessment of mouse cognition. As the phenotype of the mutant may be heterogeneous, several tests may be required to identify deficits.

Preattentive Processes

Cognitive performance is enhanced if animals can focus their attention on the most salient information in the environment [50]. Inability to filter information is thought to promote sensory overload and cognitive fragmentation [51], which are thought to contribute to cognitive impairment in several different psychotic disorders [52]. This filtering process is termed sensorimotor gating and is evaluated by PPI. This task refers to the ability of a weak stimulus to reduce the magnitude of response to a subsequently stronger stimulus (Figure 12.3). In the typical auditory PPI paradigm, a prepulse that is 4, 8, or 12 dB above the background noise is presented prior to exposure to a stronger stimulus that reliably elicits a startle response. The auditory prepulse inhibits the magnitude of the startle response and, as the intensity of the prepulse stimulus increases, inhibition of the startle response becomes more enhanced [53]. A distinct advantage in using PPI is that both animals and humans can be evaluated [43]. More importantly, PPI deficiencies are evident in many psychiatric disorders, including schizophrenia [54], thereby rendering it a replicable test for disturbances in preattentive functioning [55].

FIGURE 12.3. Prepulse inhibition.

FIGURE 12.3

Prepulse inhibition. Presentation of a weak auditory prepulse inhibits the response to a subsequent stronger stimulus. Testing is typically conducted with an interstimulus interval between prepulse and startle stimuli of 100 msec. By manipulating this (more...)

To date, most PPI studies in mice use a limited range of test parameters. In the standard test, broad-band white-noise stimuli are continually present to provide a stable background. Prepulse acoustic stimuli are presented 4 to 18 dB over this background, and they typically precede the 100- to 120-dB startle stimulus by 60 to 140 msec [54, 56]. Most studies in rats and mice, however, have used the magnitude of inhibition of PPI responses as a single response index. More recently, some investigators have examined the temporal properties and saliency of the prepulse stimuli in rats with disrupted dopaminergic tone [56, 57]. In our laboratory, the PPI paradigm consists of 20-msec prepulse acoustic stimuli that are 4, 8, or 12 dB above a 64-dB white-noise background. The prepulse precedes the 40-msec 120-dB startle stimulus by 100 msec. At the beginning of the experiment, the mouse is placed in a circular Plexiglas tube (the animal can turn around) and is acclimated to the apparatus for 5 min. Thereafter, it is given 64 test trials separated by an intertrial interval of 8 to 20 sec. Testing commences with ten startle-only trials followed by combinations of the three prepulse trials, six startle-only trials, and eight null trials (no startle or prepulse stimuli) in a pseudorandom order; testing is completed with ten startle-only trials. PPI provides a good assessment of preattentive functioning, and PPI performance can alert the investigator to deficits in sensorimotor gating that might impact performance on subsequent cognitive tests.

Attention

Attention is often defined as a heuristic concept, involving many cognitive dimensions, and it can only be evaluated through multiple test procedures [58]. Attempts have been made to classify attention into various categories including, but not limited to, reflexive attention [59, 60]; visual orientation [50]; learned orientation [61]; vigilance [62]; habituation [63]; or selective [64], sustained, and divided attention [47]. In theory, each process can be tested independently of each other; however, in practice most studies examine attention across several dimensions simultaneously [47]. For example, no single behavior executed by an animal can be labeled as “vigilance.” Instead, vigilance tests such as the continuous performance task (CPT) require the animal to maintain vigilance, orient toward the visual stimuli, scan the stimuli for change, and make a response to the proper stimulus [62]. The animal must execute all of these behaviors so that “vigilance” can be assessed. Hence, attention subsumes many processes, and these aspects of behavior cannot always be easily dissociated for study.

Orienting Responses

The simplest tests of attention involve orientation. Although tests of visual orientation are the most common [60, 63], tests have also been developed using tactile or auditory stimuli. Visuospatial orientation provides one of the most effective means to study elementary forms of information processing in animals [65]. Currently, a great deal is known about processes of visual perception [66] and the relationship of this behavior to other forms of attentional processing. Another reason for interest in orientation is due to the fact that inefficient saccades, smooth eye movements, and orienting responses have been linked to deficits in information processing for patients diagnosed with schizophrenia and schizotypical disorders [67], anhedonia [68, 69], depression and anxiety [70], obsessive-compulsive disorder (OCD) [71], attention deficit hyperactivity disorder (ADHD) [72], and Parkinson’s disease [73]. In addition, neural circuits that regulate this response are fairly well described on an anatomical [63], electrophysiological [74], and neurochemical basis [58, 59]. As orientation across different species is similar, this test can provide important insights into attentional processes.

In species other than the rodent, visual orientation utilizes fovea tracking [50, 75]. This task involves cuing the animal or human to a particular position in space prior to the presentation of a visual stimulus. When the visual stimulus is presented following the cue, information about the stimulus is processed more efficiently. This improved processing is interpreted as being due to directing attention to a specific location [50]. Unfortunately, fovea tracking cannot be easily assessed in rodents with regard to visuospatial orientation, since they have a natural propensity to orient their head or whole body toward a novel stimulus [61, 63]. Several methods have been developed in rats and mice to measure the visual orienting response. The simplest of these techniques involves bringing a single object into the field of vision and observing the ability of the animal to track the object (Figure 12.4). In our laboratory we conduct a simple screen for visual orientation by placing the mouse in an enclosed circular arena. After 2 min of habituation, a small round object (back of an infant feeding spoon) is moved horizontally at eye level around the perimeter of the arena for 12 sec. Each presentation of the spoon constitutes a test trial, and during the trial, the orienting responses of the mouse are coded at either 1- or 2-sec intervals. Orientations are defined as the mouse orienting its face toward the spoon and tracking the spoon as it moves around the perimeter of the arena, such that the animal approaches, grabs, or manipulates the spoon as it is moved. Twenty presentations of the spoon are usually sufficient to induce habituation. A variation of this rapid screen involves adding an additional ten test trials following habituation where the mouse is randomly presented with either the familiar spoon or a new object of a different color or visual pattern. Introduction of the novel object typically reinstates the orienting response toward the object, but it does not significantly alter the habituation of the mouse to the familiar object.

FIGURE 12.4. Orientation and habituation.

FIGURE 12.4

Orientation and habituation. The simplest test of attention involves orientation, which can be examined by visual tracking (top left) or various operant techniques (top right). In each case, a visual stimulus is presented over 10 to 20 trials, and the (more...)

Orienting responses to visual stimuli can also be assessed by operant techniques (Figure 12.4) [76]. In these paradigms, the rat or mouse orients toward a light cue that is paired to a particular sequence of auditory tones or clicks. The orienting response is simply defined as the animal pointing its face and snout toward the source of the visual stimulus. Habituation represents a loss of this response over testing, and it can be used as an index of novelty recognition and basic learning [76]. Moreover, the orienting response can be reinstated following habituation if a novel stimulus is introduced or paired with the familiar pattern of stimuli, creating a mismatch signal.

Modifications of the orienting response can be used to access different aspects of attention, including anticipation (which increases response efficiency during testing) and sustained attention. Anticipation is measured by delivering an auditory cue before presentation of a light cue. If the mouse learns to orient toward the location of the light cue before its illumination, then subsequent responses to lever press or nose poke as signaled by the light cue will become more efficient and rapid. Sustained attention can also be examined by increasing the time between the predictive auditory cue and illumination of a nose-poke aperture. This procedure allows the investigators to measure how long a mouse will sustain visual orientation toward the aperture in the absence of the light cue. Both anticipatory and sustained attention are important indicators of executive control and self-regulation [77, 78], and these processes may be deficient in patients diagnosed with schizophrenia [79], Tourette’s syndrome [80], and ADHD [81]. Thus, assessments of attentional control constitute an additional, but important, component of orienting-response testing for mice.

Studies using lesions in rodents or neuroimaging in primates and cats have identified a limited number of brain regions that regulate the visual orienting response. These areas include the superior colliculus, and the occipital, parietal, and frontal cortex [62–64, 82]. The superior colliculus and parietal-frontal sensorimotor cortex appear to be especially involved in the regulation of visual orientation, as lesions or brain injury of these areas produce a loss in the ability to shift visual attention [83]. The neurotransmitter systems most closely associated with forebrain control of orienting responses are acetylcholine and norepinephrine [61, 62, 84]. Interestingly, response amplitudes of noradrenergic neurons in the locus coeruleus are enhanced when the stimuli are novel rather than familiar [62].

Another brain region implicated in mediating the visual orienting response is the amygdala. While damage to the central nucleus of the amygdala does not affect spontaneous orienting responses or their habituation, this brain area appears involved in reinforced or learned orienting responses [61]. The amygdala can exert indirect influences on orientation through the dorsolateral striatum via input to the midbrain dopamine neurons [61]. Although tests of visual orientation cannot provide definitive answers concerning the mechanisms of attention, these paradigms may be useful as a first level of screening to determine whether orientation deficits are evident and whether further investigations into more specific aspects of attention and information processing are required.

Multiple-Choice Serial-Reaction Test

This test is similar to human continuous performance tasks [85] where the individual scans an array of objects that are briefly presented. Correct selection of the location where the visual target was presented results in a reward. The five-choice serial-reaction time task (5-CSRTT) is a test of attentional performance and vigilance where the mouse is required to simultaneously monitor three to five locations for the presentation of a brief visual stimulus [47] (Figure 12.5). The testing chamber typically consists of a square box where, on one side, five nose-poke apertures with lights are positioned in a horizontal line equidistant and at eye level to the mouse. On the wall directly opposite the apertures is a small opening for the delivery of food reward. The nose-poke array consists of the five apertures, where one of them is randomly illuminated. At the start of training, an aperture is typically illuminated for 20 to 30 sec, and this duration is reduced to approximately 2 to 4 sec as the mouse becomes more proficient at the task. Correct responses involve nose poking into the lighted aperture; nose poking into nonilluminated apertures results in a timeout, where the nose-poke apertures and house lights are extinguished for 5 to 10 sec beyond the duration of the 5-sec intertrial interval. The accuracy and speed of nose poking, taken as measures of attentional capacity, and the inhibition of inappropriate responses, taken as a measure of attentional control (e.g., impulsivity, perseveration), are used as core measures in this test [86].

FIGURE 12.5. Multiple-choice serial-reaction test of attention and vigilance.

FIGURE 12.5

Multiple-choice serial-reaction test of attention and vigilance. Measuring the accuracy and speed of nose poking into an illuminated aperture during the multiple-choice serial-reaction test has been used to assess attentional capacity. Inhibition of inappropriate responses (more...)

Before testing commences, the mouse is placed on food restriction and is maintained at 90% of its free-feeding weight throughout the experiment. Following food magazine training, animals are trained to nose poke for a food reward by randomly illuminating one of the nose-poke apertures. When the mice reach a criterion of 85% success over three consecutive days, serial response testing begins. The first trial of each test day is identical to that used in training, where the mice have 60 sec to make an appropriate response to the single illuminated aperture. Test trials are scored as successes or failures, and responses in each trial can be scored as impulsive/premature or perseverative. With regard to test trials, successful trials are scored when the mouse correctly nose pokes into the illuminated aperture. Failures are recorded if the animal fails to nose poke or responds by nose poking into a non-illuminated aperture. For each trial, responses are recorded as impulsive if the mouse begins nose poking before the light cue is presented or if the animal head-pokes into the food magazine before food delivery. By contrast, perseverative responses refer to behaviors that the mouse emits repeatedly within a single test trial without any reinforcement. The scoring of impulsive or perseverative responses is important during multiple-choice serial-reaction tests, as these behaviors are postulated to reflect a loss of executive control or self-regulation that is thought to contribute to deficits in attention [81].

Modifications in temporal dimensions of the stimulus cues can increase attentional load, thereby providing assessments of vigilance and sustained attention. For example, shortening the light cue to 1 sec or less will create a situation where the mouse must continuously monitor all five nose-poke apertures in order to detect the brief presentation of the cue. In addition, by extending intertrial intervals under this procedure, a maximum time limit will be reached that the mouse can sustain attention toward the five nose-poke apertures. After this time is reached, performance deteriorates. Finally, presentation of distracting stimuli such as bursts of white nose or flashing house lights either concurrently with or prior to the visual cues can be used to test attention selectivity. Animals that can focus and selectively attend to the relevant cues will exhibit higher success rates over testing compared to mice that switch attention to the distracting stimuli and, consequently, fail to notice illumination of the nose-poke aperture.

The ability of the mouse to perform various aspects of the 5-CSRTT depends upon neurological integrity of the prefrontal cortex and upon certain monoaminergic and cholinergic pathways [86]. For instance, lesions of the anterior cingulate and prelimbic cortices impair selective attention by reducing choice accuracy of the mouse. By contrast, lesions of the postgenual anterior cingulate or the infralimbic cortex promote impulsivity without impairment in other measures of attention. Prelimbic cortical lesions produce not only deficits in attention, but they also increase perseveration, particularly when the duration of the target stimulus is reduced. These findings led Dalley and colleagues [86] to suggest that attentional selectivity seems to be controlled primarily by dorsomedial areas of the prefrontal cortex, whereas ventral or lateral areas appear responsible for inhibitory control. Aside from specific brain areas, certain neurotransmitter systems also contribute to various aspects of performance. For example, lesions of ascending cholinergic pathways impair the ability of the mouse to discriminate among or between stimuli, and this debility is especially evident when the load on attention is increased by shortening the duration of the target stimulus or by the presentation of distractors [87]. Destruction of noradrenergic projections to the frontal cortex impedes accuracy of attention [62, 88], whereas depletion of both norepinephrine and dopamine from the medial pre-frontal cortex results in selective attention losses that can be corrected by amphetamine [64]. Lesions of the dorsal raphe nucleus resulting in reduced serotonergic tone in the neocortex and striatum lead to an enhancement in impulsive responses [45]. Together, these results show that certain aspects of attention are regulated through neural pathways that project to the forebrain [86].

Go/No-Go (GNG) Testing

This test is a variant of the stop-signal reaction time test that utilizes a recognition task where reaction time is analyzed (Figure 12.6). Briefly, the test can include only “go” trials or can include combinations of “go” and “no-go” trials. On a “go” trial, the animal is presented with a stimulus (e.g., light) and it must respond to this stimulus with a lever press or a nose poke. This response is rewarded. For “no-go” trials, a signal such as a tone or flashing light is presented prior to the “go” cue, and the animal must learn to withhold its response to receive reinforcement. Presentation of the “no-go” and “go” stimuli in succession within the same test trial creates a “familiarity-based conflict” and provides a context where error detection can be examined by comparing correct and incorrect responses made by the animal [89, 90].

FIGURE 12.6. Go/no-go testing.

FIGURE 12.6

Go/no-go testing. Testing is conducted in an operant chamber where a mouse is presented with either a go stimulus, where lever pressing is rewarded, or a no-go stimulus, where the animal must learn to suppress the lever-pressing response to earn a reward. (more...)

GNG testing is usually conducted in operant chambers equipped with stimulus lights positioned above two lever presses or with two illuminated nose-poke apertures. In our laboratory we have obtained the best success with mice when the levers or nose-poke apertures are situated at eye level to the mouse and mounted on either side of the food magazine. Food rewards include a magazine that dispenses sucrose pellets (20 mg) or a liquid dipper system that delivers a small volume of sweetened condensed milk. Mice are maintained on food restriction at 90% their free-feeding weights. Testing is conducted over three phases: shaping, “go training” where the mouse learns to respond to a “go signal,” and GNG testing where the animal learns to withhold its response when presented with a “no-go signal.” During shaping, mice are trained to press a lever or nose poke for a food reward. Once animals are trained to feed from a food magazine and reliably lever press or nose poke for reward under a continuous reinforcement schedule, they next receive training on the “go test” for at least five consecutive days. This test consists of 40 trials/day. Training begins with the presentation of the “go signal,” which is a light illuminated either directly over a single lever or within a nose-poke aperture. Delivery of reward occurs when the mouse responds to the appropriate cue within a certain period of time (e.g., 60 sec). The animal then has a limited amount of time to locate and consume the food reward (20 sec). Failure on either part of the task results in a time-out (10 sec) before the intertrial interval (10 sec) is imposed. Once mice reach a criterion of at least 85% success over three consecutive days, they are introduced to GNG testing. In this phase, mice receive 30 daily trials of “go testing” randomly interspersed with 30 trials of “no-go testing” over ten days. “No-go” trials are usually signified by a tone or flashing light, and the mouse must withhold its response (15 sec) to receive a food reward. If the animal responds with a lever press or nose poke during or following presentation of the “no-go” signal, the trial is terminated and recorded as a failure, and a time-out is imposed without the delivery of reward.

In a variant of the “no-go” trials, the “no-go” signal can be presented following the “go stimulus.” Under this paradigm, vigilance and impulsivity can be simultaneously assessed by varying the length of time between the “go” and “no-go” signals, and the maximum “withholding” time achieved by the animal can be assessed [91]. Another variant of the test includes lengthening the amount of time the mouse is required to withhold its response before a food reward is delivered. Animals that demonstrate difficulty in withholding a response for a certain period (e.g., 15 sec) should also be tested at shorter intervals (2 to 10 sec) to determine whether the deficit is due to an inability to withhold its response for an extended period or an inability to acquire the no-go task.

Behavioral performance in both “go” and “no-go” testing is assessed by determining the percent successful trials and the average latency to make a correct behavioral response. The data can also be analyzed with signal-detection methods [92]. Incorrect responses in GNG testing may result from impulsive or perseverative responses. Impulsive errors include repetitive lever pressing or nose poking during the intertrial interval before onset of the next trial or repeated head entry into the food magazine when the lever lights or nose-poke apertures are lit. Perseverative errors consist of the mouse repeatedly pressing a lever or nose poking into an aperture following reward delivery or persistent head entries into the food magazine following retrieval of reward.

Although the GNG test has been classified as a nonspatial recognition memory test [93], it can also be used to assess attention dysfunction and impulsivity [94]. Accordingly, GNG testing has been used to model neurological and behavioral deficits in attentional and inhibitory control found in patients with ADHD [95]. Studies in both rats [91] and humans [96] reveal that lesions of the basal forebrain and other cortical areas impair “no-go” responses while leaving “go” responses intact, suggesting that deficits observed in GNG testing could be attributable to cognitive impulsivity [81] and cognitive control [96]. Both processes appear to be regulated in the anterior cingulate cortex, a brain region attributed to conflict monitoring [97]. The frontal cortex and striatum also contribute to cognitive control [91, 98–101]. Studies in monkeys have revealed that reducing midbrain dopamine activity can impair responding on “no-go” trials and hinder the ability of the animal to switch responding between “go” and “no-go” trials, resulting in a high rate of perseverative responses during “go” trials [102]. Perseverative responses leading to deficits on “no-go” trials are also attributed to central serotonin depletion, even when levels of dopamine and norepinephrine are not appreciably changed [45]. The deficits in responding following depletion are very difficult to overcome, as this deficiency persists even when incorrect responses in “no-go” trials are punished with foot shocks [103]. Interestingly, serotonin depletion of the prefrontal cortex results in increased perseverative responses to a rewarded stimulus and an inability of the animals to withhold its response during reversal learning, even while other executive tasks remain intact [104]. Similar results have been shown in human studies with tryptophan depletion [96]. In this regard, it is important to note that cognitive perseveration and inflexibility are associated with reduced prefrontal activity in patients diagnosed with ADHD [96], schizophrenia [105], and OCD [106].

Latent Inhibition (LI)

LI is a test that examines selective attention while also addressing aspects of learning and memory. LI refers to the retardation of conditioning to a stimulus due to its prior repeated nonreinforcement [49]. LI testing consists of two phases. In the first phase, animals are divided into two groups: one receives multiple presentations of a stimulus in the test apparatus, while the other is merely exposed to the apparatus. In the second phase, the previous stimulus is associated with an aversive event. Typically, LI is evident when animals pre-exposed to the stimulus, as compared with those only exposed to the test apparatus, exhibit a delay in acquiring the conditioned response. Strategies for assessing LI include the use of appetitive behaviors with conditioned suppression [107] or the use of aversive procedures such as foot-shock avoidance [47, 49, 108] or conditioned fear [109].

In our lab, animals are divided into two different groups. One group is placed into a two-chambered shuttle apparatus and allowed to explore it for one hour. A second group is placed in the apparatus for the same period of time and is exposed to an intermittent tone and/or light as the conditioned stimulus (CS). The next day, mice are tested in shuttle avoidance, where the CS predicts foot shock. If the mouse exits the chamber during the 8-sec tone, the trial is scored as an avoidance response. If it leaves the chamber after the initial 8 sec and during the subsequent 8-sec presentation of tone and foot shock, the behavior is scored as an escape. Alternatively, if it does not exit the chamber at either time, the trial is scored as a failure. Using this system, “learning curves” can be constructed across time so that the avoidance, escape, and failure responses of the mice can be followed in detail.

In an early study, rats were administered 6-hydroxy-dopamine into the dorsal noradrenergic bundle to induce norepinephrine depletion in the forebrain [110]. LI was found to be deficient in these animals. Later studies revealed that ablation of hippocampus prevented LI and that this effect was primarily mediated through the nucleus accumbens [111]. Interestingly, N-methyl-D-aspartate (NMDA) lesions extending from the entorhinal cortex to the ventral subiculum prevented LI, and these effects were reversed by systemic administration of haloperidol [112]. Aside from this pathway, more-recent studies have revealed that lesions of the basolateral amygdala produce persistent LI and these effects, including those induced by lesions of the nucleus accumbens, are ameliorated with atypical antipsychotics [113] — possibly through antagonism of 5-HT2A receptors.

Learning and Memory

Associative Learning

Investigations of associative learning are usually conducted as paradigms of classical conditioning, where two previously noncontiguous stimuli are paired. In these tests, one stimulus is designated as the unconditioned stimulus (UCS), and presentation of the UCS elicits a reliable and measurable unconditioned response (UCR). A second stimulus is considered “neutral” at the start of testing, but when paired with the UCS, this CS elicits a conditioned response (CR). The UCS can be food or water, in which case it is appetitive conditioning, while foot shock or other noxious stimuli constitute aversive conditioning [114]. Another important paradigm for studying learning and memory is instrumental conditioning, which differs from classical conditioning in that the animal’s behavior is instrumental to the production of reward or avoidance of punishment [115]. Instrumental behaviors tend to be voluntary rather than reflexive or autonomic.

Classical and instrumental conditioning procedures are commonly used to investigate mechanisms that underlie learning and memory in rodents. Memory can be classified as either implicit (nondeclarative) or explicit (declarative), and these distinctions are based upon how information is stored and recalled (Figure 12.7) [114, 116].

FIGURE 12.7. Learning and memory.

FIGURE 12.7

Learning and memory. Processes of learning and memory in the mouse are analogous to those found in humans and nonhuman primates. Memory is classified as either explicit or implicit, with explicit memory involving the processes of encoding, storage, and (more...)

Implicit or nondeclarative memory refers to nonconscious learning that is evident through performance and does not require access to any conscious memory contents. This type of memory includes procedural habits and skills, priming, simple classical conditioning that involves the skeletal musculature and emotional responses, and nonassociative learning where the striatum, neocortex, cerebellum and amygdala, and reflex pathways mediate these respective responses [117, 118]. By comparison, explicit or declarative memories involve knowledge of discrete events, places, or facts. This information is recalled with conscious effort and is highly plastic, permitting the creation of new associations [114].

Explicit memories involve three distinct processes that include encoding, storage, and retrieval [114, 118]. Encoding pertains to the processing of information to be stored and includes input of details about a stimulus and the environment. The encoding stage has two components: acquisition and consolidation. Acquisition refers to registration of inputs in sensory buffers and preliminary analysis of this information. Consolidation may involve the reorganization of newly encoded information so that it has stronger representation in memory. Storage is the mechanism by which information is retained over time, whereas retrieval uses stored information to create a conscious representation of the event or to execute a learned motor response.

In addition to these processes, explicit or declarative memory can also categorized as immediate, short term, or long term. Immediate memory, also referred to as a sensory register [116, 119], lasts less than several seconds but has a large capacity that can receive input simultaneously from multiple sensory modalities. Short-term memory is also termed working memory and is defined as a process that recruits knowledge on a short-term basis for rehearsal, elaboration, recoding, and comparison in order to solve a current problem [116, 120]. Long-term memory is recognized as a mechanism for storing information over a prolonged period of time that can last from several hours to a lifetime [116, 120]. Although different classical or instrumental conditioning paradigms have been used with mice to examine various aspects of learning and memory, such as short- and long-term memory [33], working memory [121], consolidation [122], or emotional memory [123], it should be apparent that each paradigm requires the use of several memory components simultaneously. As will be demonstrated in the following section, the ability to examine various aspects of learning and memory within the same paradigm can provide valuable opportunities to investigate the mechanisms underlying these processes.

Avoidance Tests

Passive and active avoidance are commonly used to examine various memory functions in mice, including acquisition, short-term or working memory, consolidation, and long-term memory. Although both tests require the mouse to avoid shock, the paradigms differ from one another, in that each paradigm evaluates unique cognitive attributes of the animal (Figure 12.8).

FIGURE 12.8. Avoidance testing.

FIGURE 12.8

Avoidance testing. Avoidance tests are commonly used to examine various memory functions in mice. In passive avoidance, the mouse learns in a single trial to suppress the natural tendency to enter a darkened chamber. In one-way active avoidance, multiple (more...)

In passive avoidance, the mouse learns to avoid an adjoining chamber where the shock was previously delivered. Hence, the animal has to suppress its natural tendency to enter darkened, confining spaces. Active avoidance requires the mouse to make a proactive response to escape a chamber or area within the chamber where foot shock was previously administered [124]. Although passive avoidance constitutes one-trial learning and assesses the ability of the mouse to retain and recall information about the environment and foot shock, active-avoidance tests require multiple learning trials. For instance, in two-way shuttle avoidance, shock can be administered to either chamber, depending upon the current location of the mouse. This condition creates a conflict for the mouse, as the location where the mouse was previously shocked on one trial may become the “safe” chamber during subsequent trials. To this end, the mouse must learn to suppress the tendency to avoid a compartment where shock was previously administered and to use the CS to predict and avoid shock [125]. Active-avoidance procedures offer the opportunity to examine acquisition and consolidation within a single animal over testing [126, 127], whereas multiple animals are required in passive-avoidance experiments for the same purpose [33].

Passive Avoidance

Memories of single experiences are a rapid form of learning that provides recollections of events or places that can be adaptive to the organism [128, 129]. Passive avoidance is a test of rapid one-trial learning, where an animal is conditioned with a single aversive event and is later tested for recollection of that experience. Typically, recollection is based on the mouse’s ability to avoid a test environment where it previously received a noxious stimulus. The conditioning can be examined over very short periods (e.g., 5 min) to much longer time points (e.g., days to months) [33, 130]. By testing different mice at various time points, certain aspects of memory can be examined that include acquisition, consolidation, retention, and recall. Furthermore, extinction can also be evaluated.

Passive avoidance is typically conducted in a two-chambered apparatus in which one chamber is illuminated with a light while the adjacent chamber is in darkness (Figure 12.8). Many apparatuses are commercially available with automated gates and computerized systems that monitor the location of the mouse, making the test relatively easy and automated. Video cameras, some of which can fit directly inside the testing chambers, can be used if ethological analyses are desired. Passive-avoidance testing commences with placing the mouse into the illuminated chamber with the door separating the light and dark chamber closed. After a brief period (5 sec), the door is raised. When the mouse completely enters the darkened chamber, the door separating the chambers is closed. The latency of the mouse to enter into the darkened chamber is recorded from the moment that the door between the two chambers opens until the mouse crosses the threshold into the dark chamber with all four feet. Immediately after the door is closed, the animal receives a 2-sec scrambled foot shock (0.1- to 0.4-mA intensity). Parenthetically, we have found that some mutant strains of mice are differentially responsive to foot shock, so we always test a small subset of mice in a shock-threshold test to determine the optimal parameters for testing (see Appendix to this chapter) [33]. Thirty seconds after the mouse receives foot shock, it is removed from the dark chamber and returned to the home cage until testing, which consists of placing the mouse back into the lighted chamber and raising the door separating the lighted and darkened chambers. During the test, no shock is given and the animal is observed for 5 min. The latency to cross to the darkened chamber is used as an indicator of memory. In addition to the latency to cross, the number of crosses between the two chambers, the total time spent in each chamber, and the frequency of head pokes into the darkened chamber until the mouse actually enters the chamber can be recorded. It is also important to note freezing behavior (especially in the lighted chamber), as this behavior can interfere with typical passive avoidance.

An important consideration in the test design is the selection of the interval between conditioning and testing. The length of this interval depends upon whether the investigator wants to assess acquisition, working memory, or long-term recall. For acquisition, durations as short as 5 min can be used, whereas intervals between 30-min to several hours may be indicative of short-term memory processes [130]. Intact retention at 5 min but deteriorating or disrupted performance at 30 or 60 min can reflect inadequate memory consolidation. Long-term memory typically refers to processes invoked after 8 h following conditioning [131]; however, 24 h is more traditionally used [33, 130, 132]. We usually evaluate learning and memory in this test at 24 h and subsequently investigate other intervals based upon the findings of this test. If mutant animals demonstrate deficiencies at 24 h, then shorter intervals may be considered to exclude deficiencies in acquisition of the task or working memory. If no differences are discerned from the control animals, then longer intervals may be assessed (e.g., 48 or 96 h) or extinction can be examined by repeatedly exposing the animal to the darkened chamber. Regardless of paradigm, it should be apparent that passive avoidance cannot be presumed to evaluate only one “type” of memory.

One-Way Active Avoidance

Active avoidance can be conducted in either a two-chamber apparatus, where the mouse is required to cross to the adjoining chamber to escape shock, or in a single chamber with a single vertical pole that the mouse can jump onto when shock is administered (Figure 12.8) [124]. During training, mice are given 20 trials/day. Each test trial is separated by 20 sec, and it consists of a 10-sec tone that is followed by a 0.1-mA scrambled foot shock. The foot shock is administered for 20 sec or until the mouse escapes either into the adjoining chamber (if a two-chamber apparatus is used) or jumps onto a centrally placed pole (if a single chamber is used). On subsequent trials the latency to respond is recorded for each animal. Avoidance responses signify crossing to the other chamber or jumping onto the pole during the 10-sec tone, and escape responses occur after the tone is extinguished (e.g., 11 to 20 sec after trial initiation). Trials where mice do not avoid or escape the foot shock are failures. Daily training is continued until the mice reach a criterion that reflects learning [126]. Learning curves within and across days reveal acquisition and consolidation of avoidance, with deficient mice requiring more training to reach criterion relative to controls. Once mice reach criterion, retention of the avoidance response can be measured by testing animals at a higher criterion. Extinction is examined by exposing the mice to daily test sessions without foot shock. Controls typically learn shock avoidance within several days and require approximately 20 trials to demonstrate retention [126]. Moreover, controls retain the response in the absence of foot shock for several days during extinction. Although active avoidance allows the investigator to examine acquisition, retention, and extinction within the same animals, this test is protracted and may require several weeks to complete. As with passive avoidance, it is important to study a small subset of mice before testing in order to determine the optimal level of foot shock to use [33, 126].

Two-Way Active (Shuttle) Avoidance

Although two-way shuttle avoidance is considered a more complex cognitive task than one-way active avoidance because it evokes “conflict resolution” for the mouse, the test is generally administered over 1 to 2 days. Hence, it is more time efficient than one-way active avoidance. Conflict resolution occurs in two-way avoidance because the mouse must reenter the test chamber where foot shock was previously administered to successfully avoid foot shock on the present test trial (Figure 12.8).

Two-way shuttle avoidance begins when a mouse is placed into one side of a two-chambered shuttle apparatus. A door dividing the two chambers is opened and the mouse is allowed 5 min to explore the apparatus. Following acclimatization, testing commences over 70 trials. In the Duke Core Facility, trials are given over 70 min with a variable intertrial interval of 30 to 90 sec. Each trial begins with presentation of the CS (0.5 sec of a 72-dB 2900-Hz tone with a 5-sec 1-mA house-light) in the chamber where the mouse is located. If the mouse does not cross to the adjoining chamber after 8 to 10 sec, a scrambled foot shock is administered. Both the light and foot shock are terminated when the animal crosses to the alternate chamber or after 10 sec. Behavioral responses are coded as avoidances, escapes, or failures. Successful avoidances consist of trials where the mouse crosses to the adjoining chamber following the onset of the CS, but before the foot shock. Escapes include trials when the mouse crosses to the adjacent chamber during the foot shock. If the mouse does not cross to the other chamber within 10 sec after foot shock, the trial is considered a failure.

As with any test using foot shock, it is important to determine the level of shock to produce a forward ambulating response, without locomotor reactivity, freezing, or vocalization (see Appendix to this chapter). If the mice demonstrate an inordinate number of escapes or failures during testing, the investigator may wish to observe subsequent animals to determine whether they have locomotor problems or difficulty orienting and detecting the CS [133]. In some cases, mice may move more slowly or cautiously during CS presentation, resulting in the foot shock being administered when the animal reaches the opening to the other chamber. In this case, the mouse may associate punishment or foot shock with approaching the adjoining chamber. Increasing the avoidance phase by 1 to 2 sec, before administering the foot shock, may remedy the problem.

Besides evaluating overall levels of avoidances, escapes, and failures, examination of learning curves over the 70 test trials can reveal information regarding acquisition or loss of each response. In most studies, the two-way shuttle avoidance is used to assess learning and short-term memory within a single test (70 trials); however, this test can be easily adapted to examine long-term memory by retesting the mice 24 h after the first test. Additional time points can also be used, with intervals between the two tests as long as 6 months being reported [134].

Nonspatial Learning

Nonspatial learning typically relies upon the ability of the animal to learn relationships between different stimuli or to demonstrate flexibility during recall, and these attributes are independent of the spatial context in which initial training occurred. A discrimination between familiar and novel stimuli is used as an index of learning and memory [93]. Several methods to test this form of cognition have been developed for mice, including simple object discrimination [37, 44, 135], social transmission of food preference [136–138], or tests of matching and nonmatching stimuli [139–141]. Each of these tests exhibit several key characteristics of declarative memory, in that (a) novel information can be acquired quickly with few exposures and (b) the memory is flexible, as the acquired information can be used in situations that are quite different from the initial conditioning environment.

Object-Discrimination Test

The object-discrimination test permits rapid screening of recognition memory in mice [135]. By varying intervals between initial object exposure and the subsequent test, general assessments of short- and long-term memory can be made [131]. It should be emphasized that more-detailed studies of retention are required if deficits are detected [44, 135]. Although Tang and colleagues [135] originally habituated their animals to the test chamber for three days, we have found that exposure of the mice to the test arena for 30 min prior to object introduction is sufficient to habituate them to the test [37]. When utilizing larger test arenas, multiple days of habituation may be required before testing begins. Regardless of whether large or small arenas are used, the test area should be opaque and covered with a lid to prevent the mouse from using spatial cues outside the cage. All phases of testing are videotaped for subsequent analysis of object exploration. A potential problem with this test is that the mouse may have an innate preference for one object over another. To limit this potential confound, a small group of mice from each genotype is usually exposed to a series of different objects before instituting formal testing. Objects that elicit similar exploration times and have a similar duration of contact are used in the formal test with a new cohort of mice.

In our lab, mice are habituated to the test arena for 30 min. After this time, two identical objects are placed in opposite corners of the arena without disturbing the animal (Figure 12.9). Parenthetically, we have found that multipatterned Legos objects work well in this test. It is important that the objects be similar or smaller in size relative to the mouse, as this facilitates exploration; larger objects can promote neophobia and reduced exploration. The mouse is given 5 to 10 min to explore the objects. All incidences of contacts are coded, as well as the time spent with each object. Object contacts include sniffing and climbing on the object or attempts to manipulate the object. After this exposure, mice are returned to their home cage until retention testing. In between animals or tests, the test cage and objects are cleaned with a disinfectant to remove any odor cues. Retention testing is conducted with two Lego objects minutes to days later, where a familiar object that was used previously is paired with a novel object of approximately the same size and height. The mice are returned to the arena for 30 min, and then the familiar and novel objects are introduced to permit investigation for 5 to 10 min. The data are expressed as a preference ratio by subtracting the amount of time spent exploring the novel object from the time spent with the familiar object, divided by the total amount of time spent exploring both objects. Hence, values close to +1 indicate a preference for the familiar object, whereas negative numbers signify preference for the novel object. Typically, mice exhibit a preference for the familiar object at the 5- to 10-min interval. This suggests that acquisition of the Lego’s features is intact. Tests conducted 1 to 3 h later permit assessment of short-term or working memory, whereas tests given at 24 h reflect long-term memory [35]. It is noteworthy that preference testing can also be conducted in other sensory modalities, including olfaction or tactile sensation [137].

FIGURE 12.9. Object discrimination.

FIGURE 12.9

Object discrimination. Used as a rapid screen, the object-discrimination task allows for the examination of recognition memory in mice. By varying the interval between the initial exposure to the objects (test 1) and the preference test where the mouse (more...)

Social Transmission of Food Preference

Although this task has been available for some time for rats [142], Alcino Silva’s lab was one of the first to adapt it for mice [136]. Since that time, the test has been used as a method for studying memory and retention by several investigators [137, 138]. The advantages of the test are several-fold. First, this is an appetitive task that does not use aversive stimuli such as foot shock. Second, the behavioral measures take advantage of naturally occurring propensities of rodents to sample novel food sources and to develop food preferences from interactions with social partners. Finally, the test is devoid of spatial references [143]. Similar to the object-discrimination test, this task allows preferences of the animals to be examined at several time points, permitting assessments for acquisition processes and short- or long-term memory.

Mice are housed in pairs for several days before testing, during which time animals are placed on food restriction to reduce their body weights to 95% of their free-feeding weights. In preparation for testing, a small dot of commercially available hair dye (diluted with water) is placed on the back or rump of one mouse from each pair. This mark serves to designate the animal as the “demonstrator” in the test. The unmarked animal becomes the tester. Food is removed from mice 16 to 24 h before testing, with water available ad libitum. On the first day of testing, the demonstrator is removed from the home cage and placed into a clean mouse cage with a single bowl containing a flavored diet (Figure 12.10). After being allowed to eat for 30 min, the demonstrator is returned to the home cage, where the tester approaches the demonstrator and may interact with it for 20 min. During this time, interactions between the two animals are monitored for frequency of contacts, muzzle sniffs by the tester, and any aversive postures or responses by the demonstrator, including aversive posturing, clawing or biting the tester, or attacks. After 20 min the tester mouse is placed into a clean mouse cage with two small bowls fixed to the floor at opposite ends of the cage, creating a two-choice test. One bowl contains the diet eaten by the demonstrator; the other bowl contains a novel diet. The tester mouse is allowed to eat for 30 min, and consumption (weight) of the novel and familiar foods is recorded. The tester mice are reexamined one day later for retention using the same familiar–novel diet comparison. The preference for diet is scored in each test as the amount of novel diet eaten subtracted from the amount of familiar diet consumed and divided by the total amount eaten [137]. Positive scores reflect preferences for the demonstrator diet, suggesting the tester animal learned and retained the food preference of its demonstrator or social partner. A negative score denotes a preference for the novel diet. It is important to note that although investigators typically look for familiar preferences in the tester mice, a preference for a novel diet that is maintained over 24 h still signifies a choice that is remembered by the tester mouse. A strong preference for novelty suggests that memory is intact in the animal. However, this response suggests the tester mouse may present a novelty-seeking phenotype.

FIGURE 12.10. Social transmission of food preference.

FIGURE 12.10

Social transmission of food preference. An appetitive task that takes advantage of the naturally occurring propensities of mice to learn about novel food sources from interactions with social partners. The social transmission of food preference can be (more...)

A note should be made with regard to the selection of flavors in these tests. Our Core Facility has obtained best results using a flavored mouse mash made from equal amounts water mixed with standard mouse chow (Richmond Diet 5001, Lab Diet Inc., Richmond, IN) that is ground to create a paste that can be flavored. The paste controls spillage and permits accurate weighing of the bowls before and after testing. The most common flavors used in mouse testing are cinnamon or cocoa [44, 136, 138]. However, sampling of different flavors or scents should be performed in advance with naïve mice to ensure that flavors or scents selected for testing do not evoke any inherent bias in the mice. In our laboratory, we found that a number of mouse strains have a strong predilection to consume foods with vanilla, cocoa, and peanut butter flavors. By contrast, other mouse strains have aversions to flavors of cinnamon and similar spices, even when the concentrations of these flavors are reduced to less than 1%. In our studies with the dopamine transporter knockout mice [137], we found lemon and almond flavors to be equally attractive to the mice, and neither flavor promoted strong attraction or avoidance responses. This is an important consideration, particularly when flavors are counterbalanced across testing for pairs of animals. If an investigator finds a bimodal distribution in performance, naturally occurring biases have to be considered before final conclusions of cognitive ability can be made.

Nonspatial Transverse-Pattern Test

One of the most intriguing tests for nonspatial learning in mice is the transverse-pattern task [139, 140]. This test has advantages over the tests previously described, in that this task evaluates the ability of the mouse to make associations between cues and to learn from these representations while also applying these associations to new contingencies. One disadvantage is that the test is labor intensive and can take considerable time to complete. However, the data can be analyzed from a variety of perspectives to study cognitive abilities in mice.

In preparation for the transverse-pattern task, mice are on food restriction for at least 1 week before training starts and are maintained at approximately 95% of their free-feeding weight during testing. The mouse is first trained to dig in a small bowl that contains sand or other small objects (beads or metal ball bearings) to uncover a food reward. Shaping typically takes 5 days to complete, some mutant strains require longer training to dig with the same efficiency as wild-type controls. Testing is conducted in clean mouse cages, with two identical food bowls mounted to the floor on opposite ends of the cage. Five different odors designated as A, B, C, D, and E are used for testing and are created using various dry spices [139]; scents from sources such as talcum power, perfume, and essential oils are avoided (Figure 12.11). The spices are mixed with clean sand so that each spice constitutes only 1% of the total weight. For food rewards, 20-mg sucrose pellets for mice, or equivalent amounts of dark chocolate or dry cereal, can be used. In our laboratory we obtained the best results with small chips of dark chocolate, which are highly palatable to the mice and do not interfere or compromise the ability of the animals to discriminate the different scented sands.

FIGURE 12.11. Nonspatial transverse-pattern testing.

FIGURE 12.11

Nonspatial transverse-pattern testing. Transverse-pattern testing assesses the ability of a mouse to make associations between cues and to learn from these representations while applying the knowledge of these associations to new contingencies. The mice (more...)

Testing commences with an initial phase that is termed “premise-paired” conditioning and involves presenting the five different odors in pairs (Figure 12.11). Each pair of odors creates a two-choice test for the mouse. For premise-paired conditioning, the pairing is based on an ordered representation of the four odorant pairs, with scent A rewarded over scent B in pair 1, and scent B rewarded over scent C in pair 2, and scent C rewarded over D in pair 3, and finally scent D rewarded over E in pair 4. In the initial days of training, the mice are presented with the four pairs, with pair 1 being presented first, followed by pair 2, then pair 3, and finally pair 4. This series of trials is repeated three times on each training day for a total of 12 test trials. To ensure that spatial cues do not interfere with performance, the locations of the scents and food rewards are counterbalanced over test trials. Performance on each test trial is assessed by recording the bowl the mouse first approaches and sniffs, the bowl where the mouse subsequently digs, and whether the correct bowl is selected. Trials are scored as “correct” if the mouse picks the rewarded scent to dig in first or as a “failure” if the mouse selects the nonrewarded bowl first. As soon as the food reward is earned, the mouse is returned to its home cage.

Mice need to reach a learning criterion of at least 80% over three consecutive days before the sequence of premise pairs is randomized and interspersed with two different types of probe trials (Figure 12.11). The first probe trial, called the test of transitivity, involves scent B being rewarded over scent D. In this pair, two nonadjacent elements are selected, scents B and D, but by rewarding scent B, the ability of the mouse to perform transitive inference can be determined. The second probe trial is a novel nontransitive pairing, involving scents A and E. The nontransitive component comes from the fact that scent A is always rewarded and scent E is never rewarded. These two probe trials are essential and provide a powerful assessment for confirming whether the mice are capable of learning transitive inference using different pairs of odors.

In probe trials, mice that are deficient in transitive inference will typically demonstrate good performance when scents A and E are paired. Control groups should perform well on both probe trials. Additional confirmation of transitive inference can be assessed when two new pairs of odors are introduced, where scent W is rewarded over X, and scent Y is rewarded over Z. Typically, neither control nor mutant mice are able to solve this task, as the pairs lie outside of the established ordered-pair representation learned by the mice. Finally, once testing is completed, additional mice can be trained and tested over several days for concurrent pattern learning (Figure 12.11). In concurrent testing the pairs of scents are not overlapping as they are in transverse learning. Consequently, scent A is rewarded over B, and scent C is rewarded over scent D, and scent E is rewarded over scent F. Regardless of the order in which these paired scents are presented, odors A, C, and E are always rewarded. Moreover, discrimination of the rewarded scent from the nonrewarded scent in concurrent testing is not contingent upon which odor was rewarded in a previous test trial, as is the case in transverse testing. The concurrent test serves as a control parameter, assuring the investigator that the mouse can acquire and remember simple discriminations. If the mice are unable to make these simple discriminations, then tests of basic learning and short-term memory are required. Hence, comparison of the concurrent and transverse protocols allows the investigator to differentiate simple association learning from more demanding transverse-pattern learning.

Spatial and Contextual Learning

Spatial learning refers to the ability of the animal to learn the location of a reward [139, 144, 145]. However, in the original distinction between spatial and nonspatial learning, O’Keefe and Nadel [146] proposed that spatial learning involves the acquisition of cognitive maps that provide representation of the environment in terms of distances and directions. These maps allow animals to navigate to locations beyond their immediate perception to locate appetitive or aversive stimuli. It should be emphasized that nonspatial memory tasks do not require these maps. Cognitive maps require an intact hippocampus [146, 147], as animals with hippocampal damage exhibit abnormal exploratory activity in tests of spatial learning and experience difficulty navigating mazes to obtain food rewards [139, 148]. An analogous phenomenon is also found in humans, where hippocampal lesions result in errors in the use and organization of spatial information [147]. Although hippocampectomized patients are capable of learning new motor skills, these individuals are unable to learn new rules for applying these skills in novel or adaptive ways [149]. These findings demonstrate that the hippocampus plays a critical role in the acquisition of new information. The fact that the hippocampus is involved in explicit memory is further verified when rats are tested in multiarm mazes for both working and reference memory [150]. In these experiments, reference memory is defined by the ability of the animal to remember the location of food within the maze. By contrast, working or episodic memory refers to the ability of the mouse to use information acquired between test trials, such as which maze arms the animal had just visited and whether a food reward was found in that location. In these tests, Olton and colleagues [150] demonstrated that working memory was controlled in areas outside of the hippocampus, most notably in the frontal cortex and striatum. The relationship between reference and working memory in animals has been compared with semantic and episodic memory functions in humans [151]. This similarity between species serves to further strengthen rodent studies as tools to investigate basic mechanisms that underlie memory formation, and they also provide a foundation for understanding cognitive dysfunction in various psychiatric conditions [139].

Radial Arm Maze

The radial arm maze is used to study various aspects of learning and memory in rodents, including spatial and nonspatial attributes [145] as well as working and reference memory [152, 153]. The maze consists of an octagonal hub or central area, from which eight arms of identical size radiate outward. The original maze was elevated above the ground and had open arms so that the rat or mouse could use extramaze cues to navigate the maze [150]. More recently, intermaze cues have been employed to examine nonspatial learning [145]. In this case, specific patterns or textures are placed at the opening of each arm from the central hub or only at openings of particular arms during testing [145]. This strategy, in conjunction, with the traditional radial arm maze test, has allowed investigators to study both spatial mapping as well as working and reference memory.

In its traditional format, the radial arm maze test (also called the win-shift or win-stay paradigm) has food reward placed in some or all arms, and the animals have to remember the locations of the food or where they have just gone to retrieve the food [154, 155]. Over trials, animals learn to avoid re-entry into arms where food has been retrieved. Behavior is measured as the number of arms the animal enters before repeating an entry and the time required to retrieve all food rewards [34, 152, 156]. Animals with learning impairments typically make fewer arm visits before reentering an arm and may take longer to retrieve all food rewards. The radial arm maze has also been used to examine perseveration and impulsivity in cognitive dysfunction, which can interfere with cognitive performance [156].

Before testing in the radial arm maze, mice are subjected to daily handling and placed on food restriction to 90% of their free-feeding weight. Following five days on this regimen, animals are placed in a clear cup in the middle of the apparatus (e.g., the hub) and are given 5 min to eat Fruit-Loops food reward. Although some investigators shape the animals by scattering food along the arms of the maze [153], we have found that, once the mice learn to eat in the central hub of the maze, they have little difficulty in finding the rewards in the arms. Once the mouse has consumed food (equivalent to baiting all eight arms of the maze) reliably over three consecutive days within 300 sec, radial arm maze testing begins. In our laboratory, we utilize a protocol for mice based upon that designed for the rat by Addy and Levin [154]. At the start of the test trial, all arms or certain arms of the maze are baited, and the mouse is placed in the center of the maze inside a small cylinder. After 10 sec the cylinder is removed, allowing the mouse unimpeded access to the maze. Each arm entry is reinforced only once during the test trial. An arm entry is recorded when all four feet of the mouse cross into the arm. The animal remains in the maze until all food rewards are retrieved or until 300 sec has elapsed. Performance is assessed by the entries to repeat. In addition to this measure, the total time required for the animal to retrieve all food rewards is recorded. Aside from cognitive performance, rudimentary assessments of motivation can be made by noting the amount of food consumed. Perseveration can also be evaluated as the propensity of an animal to exit one arm and enter another arm repeatedly [156]. If behavior in the maze is video-recorded, the images can later be evaluated for the speed with which the mouse traverses the maze. High speeds with numerous errors may suggest impulsivity.

Morris Water Maze

The Morris water maze is an apparatus where mice learn to escape from water by swimming to a hidden platform located just below the surface of the water. Control animals learn this task in a relatively short time — only a few days [157, 158] or, under special training conditions, within a single day [159]. The water maze is advantageous, as it does not require food or water deprivation and takes advantage of the natural swimming behavior of the animals. Moreover, various studies have shown that an intact hippocampus is required for this task and that hippocampal-cortical pathways, as well as the dorsal striatum, play critical roles in the regulation of spatial memory in this test [160, 161].

The water-maze test requires a fairly large area of lab space where cues outside the maze can remain stable over the entire testing period. The maze uses a large circular pool of water that is made opaque by the addition of a white dye or nontoxic white poster paints. Although powdered milk can be used, the milk becomes rancid over days, and this feature increases the workload on the investigator. The behaviors of mice in the maze are video-taped with a camera mounted directly over the center of the pool. These data can be analyzed by several tracking programs such as Noldus Ethovision (Noldus Information Technology, Blacksburg, VA), SMART (San Diego Instruments, San Diego, CA), Water for Windows (HVS Image, Hampton, UK), or Wintrack (University of Zurich, Switzerland). Each program provides basic measurements, including path length, swim time, and velocity as well as a number of analyses for path-length data [162]. Lighting the pools can be tricky, but placement of the lights behind diffusers in the ceiling or having the lights mounted on the walls at water level and pointing upward can produce adequate illumination without glare from the water surface — a critical consideration when using light-based automated tracking systems. The lighting should be even and maintained at 100–600 lux when measured from the surface of the water. A moveable platform, large enough for the mouse to stand comfortably with all four feet, should rest 1 to 2 cm below the surface of the water. It is important that the platform not be too far beneath the surface of the water; otherwise the mouse will have difficultly locating the platform. Optimum water temperatures are approximately 23 to 25°C.

The pool is divided into quadrants, designated as northeast, southeast, northwest, and southwest (Figure 12.12). One quadrant is designated as the location of the hidden platform, and this position is maintained throughout testing. Release points for the mice occur at seven different locations, spaced equally apart. It is important that release points be randomized across testing, and the same release points should be used for all animals. It is also important that the investigator be hidden from view of the mice during testing, as we have found that most mice preferentially swim toward the individual who removes them from the pool rather than the platform.

FIGURE 12.12. Morris Water Maze.

FIGURE 12.12

Morris Water Maze. The Morris water maze assesses spatial learning in the mouse. The test is conducted in a large circular pool of water, where a platform has been hidden beneath the water surface in one quadrant of the pool. Mice must use spatial cues (more...)

Before commencing testing, mice should be handled daily for one week. Testing typically consists of three phases: acquisition trials, where the animal uses extramaze cues to find the location of the hidden platform; retention or probe trials, where the platform is removed and the animal uses the cues to swim to the previous location of the platform; and cued navigation tasks or “flag trials,” where a flag is placed over a movable platform to assess visual and sensorimotor function of the mice (Figure 12.12) [162]. On the day before testing, mice are trained to sit on the platform for 30 sec. They are allowed to swim freely around the maze for 60–90 sec and are then guided to the platform. This is repeated several times for each animal. If mice attempt to jump from the platform, they are returned to the platform for an additional 30 sec. Following each trial, mice are gently dried and placed back with cagemates. To avoid stressing the wet animals, it is important that the cage be kept out of drafts and, if needed, that a heating pad be set on a low setting and placed below the cage. The use of red lights or heat lamps is discouraged, as the brightness or heat from these lamps may be stressful to the animals.

On the first day of testing, the mice are released from different locations around the perimeter of the pool and allowed to swim until they find the platform or until 60 sec elapses. All mice are given four to six test trials per day, where pairs of trials are separated by an intertrial interval of 20 to 30 min. The acquisition phase of testing continues for 5 days for a total of 20 to 30 test trials. Variables used to establish learning during acquisition testing include path length (cm), the amount of time (sec) required to locate the platform, and swim velocity (cm/sec). Probe trials, where the platform is removed from the water maze, are given twice during acquisition training. We typically administer them midway and again at the end of testing. During the probe trial, the mouse is allowed to swim for 1 min with no platform present. In this time, the animal will use spatial cues to navigate and search the quadrant of the pool where the platform was previously located. Path length, swim time, and velocity are again examined, but this time they are measured for each quadrant of the pool. As the mouse acquires the task, it should demonstrate increased swim time in the quadrant on the probe trial where the platform was located previously. If no differences occur between quadrants, it is likely that the mouse has not learned to use spatial cues to find the platform. Increased swim time or path lengths in areas of the maze adjacent to the platform may indicate that the animal is confused about the relevant spatial cues or that knowledge concerning the position of the platform is still undergoing consolidation.

Although some investigators test noncued and cued navigation in the same animals, we have found that more-reliable data can be collected if naïve mice are used in both tests. On cued navigation trials, a “flag” is attached to the platform so that its location can be easily seen. We usually run 10 to 20 trials where the location of the platform is randomized across trials. This test is critical to ensure that the sensorimotor capabilities of the mice are intact. If the mice fail to swim to the visible platform, it is important that vision be examined more closely in the animals, particularly if a neurophysiological screen was not conducted. In addition, poor performance on the flag trials may necessitate examining locomotor and coordination abilities of the mice.

Mice have been shown to display distinct strategies and patterns in swimming navigation during learning [163]. Analyses of swim paths can provide some insights into these behaviors. In a detailed examination of swim paths from numerous animals, Wolfer and Lipp [163] found that in the initial phases of acquisition, mice usually adopt a pattern of thigmotaxis, or swimming along the perimeter of the maze. However, this pattern is replaced rather quickly to search behaviors referred to as scanning. These search patterns consist of the mouse engaging in swimming random paths across all quadrants of the pool or may include circling in wide loops systematically through the different areas of the pool. Scanning, in turn, will give way to a more focused search strategy, particularly after the mouse has inadvertently encountered the platform. After the mouse has acquired that task, it will swim directly to the platform. On the other hand, some mice will show random floating or will swim in very tight concentric circles that may not appear as part of any systematic search strategy. We may remove these mice from the study.

Neurological Mechanisms Underlying Learning and Memory

O’Keefe and Nadel [146] first observed that the firing rates of hippocampal cells were correlated with specific locations in the test environment. These cells were later termed “place cells” [164]. Place cells were identified rapidly upon first exposure to the novel environment [165]. Once these patterns of activity in the hippocampus were established, the firing patterns persisted; even when spatial cues were removed [166, 167]. Because place cells only fired when a specific area of the environment was encountered, it was postulated that new comprehensive maps called “place fields” were constructed in the hippocampus every time the animal entered a new environment. These data suggested that activity of hippocampal neurons was not only long lasting, but also malleable over time.

Despite numerous studies conducted to characterize the hippocampal place fields and spatial memory, it was also known that the neural activity in the hippocampus could regulate nonspatial information and sequences of behavior that occur at regular intervals [168, 169]. Hence, neural activity in the hippocampus reflects a broad spectrum of responses, with some cells encoding unique events; some encoding certain stimuli, behaviors, and locations of events; and others regulating sequences of events or specific common features across different events.

The brain areas typically associated with the hippocampal memory system involve the cerebral association cortex, the hippocampus proper, and the parahippocampal region, which includes the perirhinal and entorhinal areas. The cortical areas provide perceptual and motor information to the hippocampus through the parahippocampus [139]. The hippocampus proper is composed of several cell layers and is organized in a distinct manner that allows information to be sequentially processed through defined circuits [170]. Three circuits are well defined: the perforant pathway includes projections from the entorhinal cortex to the granule cells of the dentate gyrus; the mossy fiber pathway runs from the granule cells to the CA3 pyramidal cells; and the Schaffer collaterals represent excitatory collaterals of the CA3 region that project to CA1 pyramidal cells. Transfer of information within the hippocampus is thought to occur along two glutaminergic pathways: the mossy fibers and Schaffer collaterals [114]. These pathways are under intense investigation, as long-term potentiation (LTP) and other processes — associated with plasticity and the formation of long-term memories — are easily studied in these neurons [171]. LTP was first described in the hippocampus, where it was observed that application of high-frequency electrical stimulation or tetanus to a particular neural pathway would augment excitatory synaptic potentials when the same pathway was restimulated with a single electrical pulse [172, 173]. Initially, studies in LTP were limited to the perforant pathway [174]; however, LTP was later observed in the mossy fibers and the Schaffer collaterals [175].

One of the first demonstrations that the glutamatergic NMDA receptors were important for spatial learning and synaptic plasticity was by Morris and colleagues [29]. In these experiments, intraventricular infusion of AP5 (an NMDA receptor antagonist) or direct infusion of this drug into hippocampus resulted in loss of spatial learning by rats in the hidden-platform version of the Morris water maze. Additional pharmacological and genetic experiments continued to support the idea that NMDA receptors, particularly those in the CA1 region of hippocampus, were necessary for the acquisition of spatial memory [129]. This idea was confirmed with homozygous mutant mice that specifically lacked NMDA receptors in the CA1 region of hippocampus [18,176]. These mice were deficient only in the hidden-platform version of the Morris water maze. While multielectrode recordings in freely moving mice showed the CA1 pyramidal cells to have place-related neural activity, spatial specificity for the individual place fields was perturbed. Intriguingly, induction of LTP in the Schaffer collaterals was also blocked. Although these studies provide strong evidence for the importance of the CA1 region and NMDA receptors in spatial learning and LTP, subsequent studies reveal that nonspatial learning is also disrupted (see Fear Conditioning section below) [44, 140].

Although NMDA receptors in CA1 hippocampus appear important for the acquisition or induction of memory, recall from long-term memory seems to be dependent upon other mechanisms [129]. As individual life experiences are unique moment-to-moment occurrences, it has been proposed that similar experiences only serve to reactivate certain aspects of stored memories rather than the complete memory [177]. Hence, this process should require that only certain neurons in a particular brain region become activated. Since CA3 hippocampus contains highly modifiable synapses, this brain region was hypothesized to be important for retrieval of hippocampal-dependent memories. As anticipated, CA3 NR1 knockout mice were deficient in recall that was dependent upon pattern completion [129]. These mutants could locate the hidden platform in the Morris water maze under full-cue conditions, but when probe trails were implemented with partial visual cues present, the knockout animals were unable to locate the platform. By contrast, mice with intact CA3 NR1 receptors were capable of finding the platform under both conditions [129]. Place-cell recordings from the CA3 NR1 receptor mutants revealed that responses from these cells to the degraded cued condition were severely impaired. Collectively, results with the CA1 and CA3 NR1 receptor knockout animals suggest that NMDA receptors in the CA1 region are intimately involved in the acquisition of memory, whereas recall is highly dependent upon NMDA receptors in CA3 hippocampus [129].

Despite this evidence, other laboratories have demonstrated that areas outside of CA1 and CA3 hippocampus are also critical for learning and memory. For instance, performance in the Morris water maze requires both spatial and nonspatial learning and memory [178], and some forms of learning appear to be independent of NMDA-mediated LTP in hippocampus [129]. Other brain areas that demonstrate LTP and are implicated in the induction and maintenance of memory processes include the prefrontal cortex [179, 180] and amygdala [181]. For example, enhancement of NMDA-receptor function in NR2B transgenic mice produces improved LTP in forebrain [182]. The transgenic mice also show enhanced retention in the novel-object recognition task, the Morris water maze, and fear conditioning.

Although a number of investigators have shown that glutaminergic transmission within hippocampal-prefrontal pathways plays a key role in LTP, other neurotransmitters including dopamine [180], norepinephrine [183], and serotonin [184] also affect LTP and long-term memory. For instance, stimulation of the ventral tegmental area provokes dopamine overflow in the prefrontal cortex and leads to a long-lasting enhancement in the magnitude of LTP [185]. By contrast, depletion of cortical dopamine levels dramatically reduces LTP [180]. The importance of dopamine in LTP is further emphasized by the fact that an optimal level of dopamine D1/D5 receptor expression is necessary for adequate LTP in the hippocampus [186, 187] as well as other brain regions, including the prefrontal cortex [188] and striatum [189]. Noradrenergic contributions to synaptic plasticity and LTP in both cortical and hippocampal areas have also been documented [190, 191]. Noradrenergic pathways, which originate in the locus coeruleus and innervate both the cerebral cortex and hippocampus, have been found to modulate glutamatergic activity and synaptic plasticity at perforant synapses in dentate gyrus [190], at mossy fiber synapses [192], and in CA1 hippocampus [193, 194]. In addition to catecholamines, serotonin also controls synaptic plasticity [195, 196]. Acute systemic administration of a serotonin reuptake inhibitor (fluvoxamine) increased the efficacy of synaptic transmission in the hippocampal-prefrontal cortical pathway [195]. When the rats were treated for 21 days with fluvoxamine, a marked enhancement in LTP was observed. Alternatively, destruction of the serotonin neurons in this pathway also enhanced LTP [196]. Both effects were attributed to disinhibition of the serotonin 1A receptor that regulates both NMDA-receptor function and LTP in the prefrontal cortex and hippocampus [197]. These findings are intriguing because they suggest that regulation of learning and memory involves mechanisms beyond glutamatergic control. In this regard, the atypical antipsychotic clozapine, which can bind to norepinephrine, dopamine D4, and serotonin receptors, has been found to reverse stress-induced impairments in LTP and cognitive functioning [180]. These studies provide critical links between mouse models of cognitive dysfunction and analogous symptoms observed in human psychiatric patients, suggesting an important role for these brain areas in the pathophysiology of various psychiatric conditions.

Conditioned Emotional Responses

The conditioned fear response has become a common and powerful paradigm with which to study the neurological basis of emotional responses as well as learning and memory. This Pavlovian paradigm involves exposing the animal to a neutral conditioning stimulus (CS) and pairing this to an aversive unconditioned stimulus (UCS). Parenthetically, the CS is usually a light or tone, and it usually also cues within the same chamber where the animal was conditioned. After pairing the CS and UCS, the CS alone often elicits a defensive or “fearful” response that may involve immobility or freezing behavior.

Conditioned Taste Aversion (CTA)

CTA was discovered when investigators realized that irradiated rats avoided solutions or food that had been present during radiation treatments [198]. When rats encountered a novel taste (the CS) and this was followed by transient gastrointestinal distress caused by low-dose radiation (the UCS), CTA developed. This response results in a diminished intake of saccharin upon subsequent presentation. Later studies found that CTA could develop following exposure to a variety of other illness-producing agents, including chemotherapeutic agents, high doses of apomorphine or amphetamine, and lithium chloride [199]. For CTA to develop, the animal must be able to detect the CS; it must be able to become ill from UCS exposure; it must be able to form an association between the US and CS; and, finally, it must be able to avoid the CS.

CTA is a relatively simple test to conduct, and it typically requires two days of combined training and testing. Mice are placed on food restriction prior to testing to ensure that they will consume an adequate amount of the novel food during conditioning and to guarantee that an association between the CS and UCS will develop. Alternatively, water restriction can be used so that lithium chloride treatment can be paired with the consumption of a saccharin solution. In our laboratory we have found that both procedures work equally well with mice. The advantage of using a novel flavored food is that it is highly palatable to the mice, the amount of food consumed is easy to measure, and testing can be conducted over a brief period of time. By comparison, saccharin-flavored solutions typically require longer consumption periods to ensure that an adequate amount of the solution has been consumed. In addition, when using a liquid CS, intake can take several days to stabilize, and analyses of fluid intake typically require more elaborate and precise measurements, such as those provided by eating and drinking chambers available for mice (see Columbus Instruments, Columbus, OH). Readers interested in using saccharin-flavored water in CTA for mice should consult Cannon and colleagues [200].

Mice are individually housed the day before testing and placed on food restriction. On the test day, each mouse is placed into a clean test cage with a flavored diet located in a small feeding dish mounted in the center of the cage (Figure 12.13). The ground chow can be flavored with either 1% vanilla or almond extract and sweetened with a 0.25-M saccharin solution. Mice should be counterbalanced across flavors, so that half the animals are conditioned with vanilla-flavored chow and the other half with almond-flavored chow. The bowl with flavored diet is weighed before conditioning and again after 30 and 60 min of exposure. Because rodents typically restrict their initial consumption of novel food sources [201], it is important to weigh the bowl at both 30 and 60 min, allowing the mouse to consume an adequate amount of flavored diet. Mice on food restriction or food deprived the day before testing typically consume 0.3 to 1.0 g of food during the 1-h free-feeding period. After 1 h the mice are removed from the test chamber and injected with either 0.15 M lithium chloride or sterile water. The mice are returned to their home cage, maintained on food restriction, and tested 24 h later for retention. CTA testing involves a two-choice test between the two flavored chows, almond and vanilla. Mice conditioned to almond may be expected to prefer vanilla and those conditioned to vanilla should prefer almond. If control animals fail to demonstrate a preference between the two flavored chows, then several factors should be investigated, including increasing the lithium dose or increasing the saccharin concentration relative to the flavoring concentration to ensure that the animals are cued to flavor and not sweetness of the diet. It is also important that the mice be naïve, with no previous exposure to either almond or vanilla flavoring/scents or saccharin solutions before testing. Any of these experiences can create latent inhibition in the mice and weaken the CS-UCS association during conditioning [202].

FIGURE 12.13. Conditioned taste aversion.

FIGURE 12.13

Conditioned taste aversion. A simple and rapid test to assess the ability of a mouse to develop a conditioned emotional response. Following food restriction, the mice are allowed to consume flavored chow. Immediately following consumption, the animals (more...)

A number of research studies have shown that the amygdala appears to be an important brain region for regulation and expression of CTA [199]. Although rats with lesions of the medial or central nucleus of the amygdala fail to demonstrate deficiencies in CTA, lesions of the basolateral amygdaloid (BLA) nucleus induce profound disruptions of CTA. The effect of BLA lesions has also been attributed to a reduction in neophobia. The BLA receives afferent information from all sensory modalities and relays this information to the central nucleus. The central nucleus is reciprocally connected to the hypothalamus and nucleus accumbens through stria terminalis and to the dorsal medial nucleus of the thalamus, the rostral cingulate cortex, orbital frontal cortex, and brain stem through the amygdalofugal pathway [203]. Given that lesions of the central nucleus fail to disrupt CTA, it can be assumed that the locus of control for this behavior rests within the BLA [199].

Fear-Potentiated Startle (FPS)

FPS is a test of Pavlovian conditioned fear where a reflexive acoustic startle response can be augmented when a startle stimulus is presented with an aversive stimulus that elicits a fear response [204, 205]. FPS has a number of features that make it attractive to investigators [206]. For instance, the reflexive startle response requires no learning on the part of the animal. The test is fully automated, and the potentiated startle response is long lasting and allows examination of long-term memory and extinction [207]. Unlike other tests of conditioned emotional responses, FPS can be examined following fear conditioning, permitting the investigator multiple opportunities to examine learning and memory under different paradigms [208]. Finally, the neural circuits that regulate emotional learning and memory are well characterized from the molecular to neurological levels [209].

Typically, FPS is conducted across four days (Figure 12.14). On each day, mice are acclimated to the chambers for 5 min before testing. On the first day, baseline startle responses are measured across a range of acoustic startle stimuli. On the next day, startle responses are paired with the CS. On the third day, mice are conditioned to the CS and scrambled electric shock, and on the fourth day, potentiation of the startle response to the CS is evaluated without shock.

FIGURE 12.14. Fear-potentiated startle.

FIGURE 12.14

Fear-potentiated startle. A Pavlovian test of conditioned fear that utilizes the reflexive acoustic-startle response. Mice are examined for baseline startle responses on the first day. On the second day, animals are presented the startle stimuli and the (more...)

Baseline testing on day 1 involves administering 40-msec bursts of white noise at several different intensities (100, 105, and 110 dB). Mice should demonstrate moderate startle responses to these stimuli. If the startle responses are too high (e.g., over 800 mA), mice are re-examined at lower intensities beginning at 90 dB. High baseline startle responses can result in a “ceiling” effect where potentiation of the response is more difficult to measure on subsequent test days. It is also important to evaluate mice on only 10 to 15 trials, where each stimulus is presented three to five times each, because the mice can habituate to these acoustic stimuli if they are administered too frequently [210]. On day 2, mice are placed back into the test chambers and given nine trials of baseline startle stimuli (three presentations/intensity). They are then presented with 18 test trials with these same acoustic stimuli (six presentations/intensity). On half the trials, the startle stimuli are administered immediately following a 30-sec, 12-kHz, 70-dB tone (CS); on the other half no tone is given. The tone+startle trials are interspersed in a pseudorandom order with the startle-only trials. The magnitude of response to the CS (e.g., preconditioning potentiated response) is calculated by subtracting the response to startle-stimulus-only trials from the tone+startle-stimulus trials, dividing this number by the startle-stimulus-only responses, and multiplying the final score by 100. Positive values indicate that the tone augments the baseline startle response, whereas negative values suggest a reduction in startle response to the tone. If an increase or decrease of the response exceeds 15%, then the animals will have to be re-examined under various intensities of tone and white-noise startle stimuli until more desirable levels are obtained.

On day 3, the tone is presented and, at its termination, a 0.25-sec 0.3- to 0.4-mA scrambled shock is administered. The mice receive ten CS-UCS pairings, separated by an intertrial interval of 90 to 180 sec. Twenty-four hours later, the mice are tested for FPS under the same procedures used for day 2. Potentiation is measured by the same formula used to calculate the preconditioning potentiated startle. For animals that acquire FPS, the percent potentiation should be increased following conditioning compared with values obtained before conditioning. Parenthetically, C57BL/6 mice typically demonstrate FPS of 50 to 100% when postconditioned potentiation is compared with preconditioned responses [206]. In our laboratory we have generally found that control groups of unaffected WT mice or inbred C57BL/6 mice produce similar levels of FPS.

The FPS paradigm has a distinctive advantage over other tests of conditioned emotional memory in that it can be evaluated in humans of all ages with parameters similar to those used for rodents [211, 212]. Adult human patients diagnosed with posttraumatic stress disorder, depression, or bipolar disorder, and children with anxiety or temperament abnormalities, demonstrate enhanced and abnormally persistent FPS responses [213–215]. In addition, FPS in animals has been proposed to parallel the development of pathological fears and phobias in humans, where persistent and exaggerated responses to fear-provoking stimuli have been documented [216]. Anxiolytics block FPS in rodents, whereas anxiogenics can potentiate the response [217]. FPS is quite sensitive to drugs known to modulate states of fear and anxiety in both rodents and humans, including norepinephrine antagonists, benzodiazepines, opioids, and atypical anxiolytics [218, 219].

Although hippocampal or amygdala lesions interfere with expression of FPS [220, 221], the amygdala is considered the key site for FPS regulation [222]. For instance, the central and lateral amygdala are stimulated in response to the CS, and this activation appears to promote enhanced and prolonged neural responsiveness [223]. Besides these regions, the BLA also appears to be involved in FPS, as pharmacological lesions of the pathway between the central and basolateral nucleus impair expression of FPS following conditioning [207]. Moreover, acquisition of the conditioned emotional response for FPS is blocked when competitive NMDA antagonists are injected directly into the BLA; these agents also disrupt or prolong extinction of FPS. Hence, these findings indicate that the various regions of the amygdala serve different functions in FPS and that the basolateral amygdala is critical for the formation and plasticity of emotional memories that underlie conditioned emotional responses. For these reasons, FPS testing can provide important insights into the neural mechanisms that underlie associations between fear and anxiety found in human anxiety disorders [185, 209].

Fear Conditioning

The idea behind fear conditioning is that a fearful experience establishes an emotional memory that can result in long-term behavioral changes and, in some cases, these changes can become part of the permanent behavioral repertoire of the individual [224, 225]. In this paradigm, mice are conditioned by pairing a tone (CS) with foot shock (UCS). The animals are later examined in two tests; one evaluates contextual fear and the other examines fear responses elicited by the CS (cued fear conditioning). Fear in both tests is signified by immobility or freezing behavior (Figure 12.15). This response is a direct reflection of the conditioned emotional response in animals [226]. By examining freezing behavior in the different test conditions, dual mechanisms underlying conditional emotional memory can be examined. Freezing during the context test is attributed to hippocampal or temporal lobe processes. Deficits in freezing during the context and cued tests are indicative of amygdala dysfunction.

FIGURE 12.15. Fear conditioning.

FIGURE 12.15

Fear conditioning. In Pavlovian fear conditioning, a fearful experience establishes a memory that can result in long-term behavioral changes. On the first day, mice are placed into a chamber and conditioned with a tone paired with foot shock. On the second day, (more...)

The strengths of the fear-conditioning paradigm are several-fold. For instance, conditioning only requires a single session. Additionally, the stimuli are under direct control of the investigator, and the behavioral responses have been operationally defined, validated, and are simple to measure. Further, the neural substrates for fear conditioning in animals have been identified. It is noteworthy that abnormalities in the hippocampus and amygdala have been implicated in psychopathologies such as schizophrenia [227] and anxiety disorders [228, 229], including obsessive-compulsive disorder [230] and posttraumatic stress disorder [231–233].

Fear conditioning and testing can be conducted over two to three days, depending upon whether the same animals are used for the context and cued tests or whether different groups of mice are used in each test. In practice, we have found no differences between these two conditioning protocols. Typically, fear-conditioning testing is conducted either in a single chamber where the contextual features of the apparatus (e.g., lighting, floor texture, wall shape and texture, and visual cues) can be altered [13] or in two to three different chambers [231]. Behavioral responses are videotaped for later analyses with ethological scoring programs such as the Noldus Observer (Noldus Technologies, Blacksburg, VA) or are scored live by trained observers. Automated fear-conditioning chambers are also available, which permit simultaneous videotaping of mice with an automated threshold-detection system that monitors movements of the mice (Med-Associates, St. Albans, VT). The investigators can set detection thresholds so that freezing behaviors fall beneath one threshold and reactive behaviors such as jumping or running register above another. Activity between the two thresholds may constitute grooming, sitting, or low-intensity exploratory activity. When using these automated systems for fear conditioning, it is critical that the investigator first observe a pilot group of animals for freezing and general exploratory behaviors so that thresholds can be set to accurately differentiate these behaviors. In our laboratory we use both ethological and automated measures simultaneously. More recently, a fully integrated system has become available (Med-Associates, St. Albans, VT) that controls stimuli presented to the mice and records all behavioral responses through threshold-detection systems and video analyses.

During testing, particular precautions must be taken so that the observed emotional responses can be attributed directly to conditioning and not other influences. Novel scents can be powerful manipulators of behavior in rodents and, under particular circumstances, can provoke freezing behaviors [234]. Hence, it is important that the test chambers be cleaned adequately between animal testing. We use unscented Anlage spray (VWR International, West Chester, PA) or NPD-128 (VWR). The use of alcohol or ammonia-based cleaners between testing is discouraged, as these agents produce lingering odors that can be distracting to the animals during subsequent tests. Animals should be maintained in group housing before testing, as it has been shown that isolation can induce abnormal behaviors and interfere with attention [235] and learning [236]. During conditioning and testing, it is important that the mice be housed or maintained in rooms separate from the test room. If animals are to be evaluated in both contextual and cued fear conditioning, then modes of transit to the test rooms should be changed so that subtle but salient cues are not transmitted to the animal prior to testing. The investigator should also wear different lab coats and gloves for conditioning and testing.

In our lab, the mouse is placed in the test chamber for 2 min. After this time, a 30-sec, 65-dB, 2900-Hz tone is given, and 2 sec before termination of the tone, a 0.4-mA scrambled foot shock is administered where the tone and shock terminate simultaneously. The mouse remains in the apparatus for 30 sec before removal to the home cage. Parenthetically, while we find a single conditioning trial is often sufficient for conditioning, two to three CS-UCS pairings can provide more robust results when the pairings are separated by 30 to 60 sec. During the entire conditioning session, all incidences of spontaneous freezing behaviors are recorded. It may also be informative to observe whether the mice orient to the CS and whether they respond to the UCS during conditioning (e.g., running, vocalizing, or jumping). Mice that do not show these responses may have hearing loss [133] and be insensitive to the level of foot shock (see Appendix to this chapter).

Following conditioning, the mouse is returned to its home cage and tested 24 h later. Retention is measured in both the context and cued tests (Figure 12.15). For context testing, the mouse is returned to the original conditioning chamber and allowed 5 min of free exploration in the absence of tone. The next day the animal is evaluated in cued fear conditioning. Cued testing involves placing the mouse in a new test chamber or in the conditioning chamber where previous sensory cues have been disguised with novel floor textures, changes in wall patterns or colors, etc. In the cued test, the animal is allowed to explore the new surroundings for 2 min, after which the CS is presented for 3 min. In each test, freezing behavior is scored and expressed as the total sec of freezing, the percent test time freezing, or the percent time spent immobile per min [13]. The latter option is advantageous in that the pattern of behavior can be examined across testing for delays in freezing following exposure to the chamber or tone, or for early extinction of the behavior. Titration of the interval between conditioning and testing can be used to examine the acquisition and consolidation of emotional memory [181]. Alternatively, strength of the emotional memory can be evaluated through repeated apparatus exposure to follow extinction [237].

In fear conditioning two CS exist; one related to the contextual fear and the other associated with the auditory cue. As the contextual CS comprises the testing environment, the CS is more generalized and is present continuously throughout conditioning and testing. By comparison, the cued CS is usually restricted to a single sensory modality (e.g., tone) that is precisely timed to coincide with the presentation of the foot shock. Due to these distinctions, the contextual CS is more plastic and readily amenable to extinction compared with the cued CS. These observations suggested to Phillips and LeDoux [238] that different neural pathways control the expression of conditioned emotional responses in these two forms of fear conditioning. While the amygdala is critical for expression of conditioned fear in both the cued and contextual tests, the hippocampus is involved in contextual fear. Lesion studies have confirmed these hypotheses [238].

Conditioned fear is mediated by brain areas that can detect, process, and direct behavioral responses to perceived danger [225, 226]. Various investigators have consistently demonstrated that the amygdala is critical for the induction, BLA, and maintenance of conditioned fear [181, 239]. More specifically, the basolateral is a locus of sensory input that is proposed to be the location where the CS and UCS are initially associated. Neurons in the ventral hippocampus, subiculum, and CA1 region project to BLA, and damage to these areas can interfere with the development of contextual fear conditioning [224]. In addition, the central nucleus of the amygdala sends projections to the hypothalamus and brainstem to support the behavioral responses. Although lesions to the central nucleus can disrupt the expression of conditioned fear responses, damage to the specific projection areas can interrupt individual conditioned responses. For example, lesions to the periaqueductal gray can perturb freezing behavior while leaving blood pressure unaffected. Additionally, disruptions to the stria terminalis also interfere with conditioned release of adrenocorticotropin while leaving freezing and blood pressure responses intact [224].

Both glutamate and dopamine have been shown to be important for fear conditioning. For instance, NMDA receptors in the dorsal hippocampus and CA1 region play an important role in controlling freezing responses in contextual, but not cued, fear conditioning [44, 240]. Dopamine is also an important regulator of fear conditioning [225]. Dopamine D1 and D2 receptors are highly expressed in the amygdala [241]. Fear-arousing stimuli lead to the activation of dopamine neurons and the enhancement of dopamine neurotransmission in the amygdala [242]. Specific roles of the dopamine D1 and D2 receptors in fear conditioning have also been elucidated with mutant mice and drugs. When the D1 receptor mutants are examined in fear conditioning, they exhibit conditioned fear responses but fail to show extinction when repeatedly reexposed to the conditioning context [130]. Although D2 receptor homozygous mutants have not been examined in fear conditioning, administration of a selective D2 receptor antagonist (raclopride) into the amygdala impairs the acquisition and retention of the conditioned emotional response [243]. Bilateral intra-amygdala infusion of another D2 receptor antagonist (eticlopride) also disrupts formation and consolidation of emotional memory [244]. The roles of both the dopamine and glutamate in conditioned fear are particularly interesting because aberrations in these neurotransmitter systems are implicated in the pathologies of several psychiatric disorders where inappropriate fear responses are reported [225].

Summary and Comments

The purpose of this chapter was to provide a general overview of tests designed to evaluate cognitive function in mice. An ancillary goal was to make the researcher aware that neurological and psychiatric dysfunction can also perturb cognition and that these complex behaviors have symbiosis. Given these relationships, it is important to evaluate mice on multiple dimensions of behavior to identify their phenotype. In cognitive testing, multiple behavioral and physiological deficiencies can confound cognitive performance. Finally, since the genetic background of mice can significantly influence behavior [245, 246], it is critical that the appropriate controls be included in the study so that any behavioral dysfunction can be more clearly ascribed to the gene(s) under study rather than representing other influences.

To date, animal models that use mutant mice have provided new insights into behavioral function. Although these models have given invaluable information, it is important to remember that these animals only provide approximations of the symptomologies and deficiencies of human patients. Nevertheless, subjecting mice to a comprehensive battery of tests provides a better framework for understanding not only the overall behavioral phenotype of the mutant, but also for more fully recognizing the limitations of the specific animal model. Despite this precaution, it should be emphasized that the goal of animal research is not to mimic precisely the human diseases or disorders under study, but to provide greater insights into basic genetic and molecular mechanisms involved in expression of the behavior. Once these mechanisms are better understood, new therapeutic strategies can be explored and developed for treating human patients.

Appendix: Shock-Threshold Testing

To evaluate sensitivity to foot shock, animals are exposed to different intensities of scrambled foot shock, and their behavioral responses are videotaped. In our laboratory, we use seven different intensities of foot shock (0, 0.05, 0.1, 0.15, 0.2, 0.3, and 0.4 mA) and present them in a random order, where each intensity is presented three times over 2 sec. Behavioral responses are scored by hand or with a computerized behavioral scoring program (Noldus Observer, Noldus Information Technology, Leesburg, VA). Responses are placed on a rating scale, with the lowest level of response scored as zero, indicating no overt response to the foot shock. Low-level responses include freezing, face wiping or self-grooming, shaking, or rapid forward departures; whereas moderate responses include tail rattling or retreating from foot shock. Moderately reactive responses include kicking, vocalization, and locomotor reactivity such as darting and leaping. The highest level of response includes jumping against the walls or ceiling of the chamber. Behavioral scores are summed based on the type of response and analyzed as a function of shock intensity. For behavioral conditioning, we select a level of foot shock that promotes only moderate responses. Foot shocks that induce continuous vocalizations, darting, or jumping may traumatize the animal and lead to inconclusive results.

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Copyright © 2006, Taylor & Francis Group, LLC.
Bookshelf ID: NBK2527PMID: 21204369

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