Behavioral and Cognitive Testing Procedures in Animal Models of Epilepsy

Chapter 13

Behavioral and Cognitive Testing Procedures in Animal Models of Epilepsy Andrey M. Mazarati Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States

IMPAIRMENTS IN LEARNING, MEMORY, AND COGNITION Spatial Cognition and Memory Behavioral assays are summarized in Table 13.1.

Morris Water Maze Morris water maze (MWM) is by far the most widely used assay for analyzing spatial learning and memory in rodents (Vorhees and Williams, 2006, 2014). The apparatus is a large circular tank filled with water, divided in four virtual quadrants, one of which contains a submerged platform. When swimming, the animal is expected to learn the location of the platform by using spatial cues, and ultimately to escape by climbing on the platform. The animal engages several strategies for learning the platform location, such as a spatial strategy (i.e., using distal cues as points of reference to locate itself in space; this can be a large distinctly shaped sign placed on the wall in a fixed location, with the reference to the platform), a taxic strategy (i.e., using visual cues for reaching the platform), and a praxic strategy (i.e., remembering the movements needed to reach the platform) (Brandeis et al., 1989). After initial habituation, consisting of placing the animal for 2 min in the tank without the platform, the test begins. The first is the acquisition phase, which lasts 3–7 days, depending on the experimental goals and set cutoff conditions. On each day, 4–6 trials are performed every 5–15 min. For the trial, the animal is placed in the tank, each time with randomly assigned starting points. If the animal fails to locate the platform within 60–90 s, it is manually guided to the platform. After spending 10– 20 s on the platform, the animal is removed. As the training progresses, the time between placing animals in the tank and it reaching the platform shortens progressively (Fig. 13.1A, i, ii). During the acquisition phase, the speed of learning the location of the platform (i.e., the number of trials required for optimal performance) serves as a measure of spatial learning. Models of Seizures and Epilepsy. http://dx.doi.org/10.1016/B978-0-12-804066-9.00013-4 Copyright © 2017 Elsevier Inc. All rights reserved.

The next phase is probe trial, assessing spatial memory. For this, the platform is removed and the animal is allowed to swim for 2 min. The time spent in the target quadrant (i.e., where the platform was located) versus other three quadrants is calculated, and the longer the time, the better the memory (Fig. 13.1A, iii). Probe test may be followed by retraining on the next day by repeating the acquisition procedure that allows additional evaluation of spatial learning. Normally, the number of trials to relearn the platform location during retraining is smaller than the number of trials during acquisition. Another optional extension to the test allows examining learning flexibility, whereby upon learning the initial platform location, the platform is placed in the different quadrant, and the animals’ ability to learn the new location is analyzed. If animals perform adequately during the acquisition and probe trials, but fail the reversal-learning phase, this may suggest the presence of autism-like and/or schizophrenialike impairments, and can supplement respective tests (see respective sections). Given significant test duration, spontaneous seizures represent a major confounding factor. Not only seizures developing during trials are disruptive, but also seizures occurring between the tests complicate discerning between chronic impairments in spatial learning and memory, and postictal amnesia. Next, as basic swimming ability is a prerequisite, measuring swimming speed during habituation provides a clue as to whether basic swimming ability is preserved. Depending on the degree of the swimming speed reduction, the latter can be either factored into the corrected outcome measures, or should serve as a contraindication for the test all together. Furthermore, intact coordination and balance are critical for adequate performance. Even slight impairments may increase thigmotaxis (i.e., the tendency to swim along, and stay in contact with the wall), and thus increase the time to reach the platform. A factor potentially producing false-positive result, particularly during the acquisition phase (i.e., failure to learn the platform location) is the presence of comorbid depression-like impairments, namely hopelessness/despair that would interfere with 181

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TABLE 13.1 Tests for Impairments in Learning and Memory Test Description

Examined Symptom

Signs of Impairment

Morris water maze

Impaired spatial learning and memory

Increased number of trials to learn platform location (acquisition) Shorter time spent in the target quadrant (probe)

Barnes maze

Increased number of trials to learn escape hole (acquisition) Shorter time spent near the target hole (probe)

Eight-arm radial maze

Impaired working memory

Increased number of trials to learn the location of a baited arm

Novel object recognition

Object and episodic memory

Similar time spent exploring familiar and novel objects

“What Where Which” object recognition Contextual and cued fear conditioning Social transmission of food preference

Similar time spent exploring familiar and novel objects in the context setting Contextual memory

Reduced freezing time upon presentation of conditioned context and/ or stimulus Equal time spent engaging with unfamiliar and familiar conspecifics

forming adequate escape strategy (see Forced Swimming Test later). Discriminating between the two may be challenging if at all possible.

Barnes Maze Similarly, to MWM, the Barnes maze examines spatial learning and memory, but its physiological basis is different. Whereas MWM relies on the fact that animals do not

like, the Barnes maze explores animals disliking bright ­illuminated areas, and instead favoring dark confined spaces (Harrison et al., 2006; Vorhees and Williams, 2014). The apparatus is a large circular surface with 20 holes located around the circumference, placed under bright overhead lighting. Only one of the holes allows for escape, and contains an escape box underneath. Visual cues are placed around the apparatus. For the probe trial, the access to the escape hole is blocked. Similar to MWM, the animal

FIGURE 13.1  Memory tests. (A) Morris Water Maze. The animal’s movement in the tank is illustrated by the curved trace line. (Ai,ii) During the acquisition, as the animal learns the location of the submerged platform (black square), the path/time between the placement of the animal in the tank and locating the platform shortens. (iii) During the probe, trial the animal spends more time in the quadrant where the platform was located. Star denotes a visual cue. (B) “WWWhich” object recognition test. (Bi,ii) The animal is successively exposed to two contexts (exposure 1: open field with mesh floor, a cube on the left, and a cylinder on the right; exposure 2: open field with smooth floor, the cylinder on the left and the cube on the right; star denotes a visual cue). During the two exposures, the animal spends equal time exploring both objects. (Biii) After a delay, the animal is placed in the open field similar to the one from the exposure 1, but with two cubes. The cube on the right is familiar in the shape but novel in the context; hence, the animal spends more time exploring it than the cube on the left.

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is ­expected to learn the location of the hole leading to the escape box. The phases (i.e., habituation, acquisition, and probe; for the latter the access to the escape box is blocked) are run and analyzed similarly to MWM. An advantage of the Barnes maze over MWM is that physical requirements (i.e., balance, coordination, and moving speed) are less strict, and hence the test can be performed in animals which are unfit for MWM. However, as the motivational strength of aversive stimuli is much lower in the Barnes maze than in MWM, lack of motivation (e.g., associated with depression) is more likely to produce falsepositive outcome than MWM.

Working Memory Eight-Arm Radial Maze The test exploits the inherent ability of rodents to devise optimal exploratory strategies for foraging. Several versions of the test allow discerning between working and reference memory (Olton et al., 1977; Vorhees and Williams, 2014). The apparatus consists of eight equidistantly spaced arms radiating from the central platform. The latter is separated from the arms by guillotine doors that close arms individually. Prior to and during the test, the animals are on food restriction, with the goal of reducing their weight to 85% of basal. The test is preceded by one week of habituation, during which the animal is placed in the center of the apparatus, with all doors open, and food scattered along the arms (during first 3 days), and then only placed at the ends of the arms. The goal is that the animal familiarizes with all eight arms. The test proper starts with all eight arms baited with food pellets. The animal is placed in the center of the apparatus with all doors open. Once the animal enters one of the arms, the remaining seven arms are closed while the animal is consuming the food in the visited arm. Upon the animal’s return to the center, the visited arm is closed. The animal remains confined to the central platform for several minutes, then all doors are opened, and the sequence is repeated. The session continues until food from all arms is retrieved. The sessions are repeated every day until the desired accuracy is reached, with a general cutoff of 15 days. The accuracy is defined as the animal not entering previously visited arms, and is typically set at >85%. The number of trials/days required for the animal to reach the set accuracy is used as a measure of working memory. In a version of the test that allows discriminating between working and reference memory, only four out of eight arms are baited (the same four arms are baited throughout the test). Here, reentries in baited arms serve as indicators of errors in working memory, while entries into unbaited arms point to errors in reference memory. Similarly to other tests relying on positive reinforcement, the presence of anhedonia may produce false-­negative

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result. Also, in models with spontaneous seizures, it may be difficult to discern between memory deficits characteristic of chronic epileptic state and those associated with postictal syndrome.

Object Recognition and Episodic Memory Object Recognition The test relies on rodents’ inherent curiosity, manifested as a preferential exploration of newly encountered objects versus familiar ones (Ennaceur and Delacour, 1988). The test is typically performed in the open field. After 5 min of the animal’s habituation, two objects are placed in two quadrants along the diagonal axis; the quadrants are offcenter, nonadjacent to the walls, as well as between themselves. The objects are of two distinct shapes (e.g., cube and pyramid, cube and cylinder), but of approximately similar sizes. For the acquisition phase, the animal is introduced into the open field, is allowed to explore the objects for 10 min and then removed. For the retrieval phase, one of the objects is replaced with a third differently shaped unfamiliar object, while the other object is the same as during the acquisition (e.g., cube–pyramid for the acquisition and cube–cylinder for the retrieval). The interval between the acquisition and the retrieval varies, depending on the goals. When evaluating short-term memory, that is, the one driven by rhinal and perirhinal cortices (Barker et al., 2007), the interphase interval lasts 5–10 min; for studying long-term memory (in which the hippocampus is involved; Hammond et al., 2004), the interval is several hours, and up to 24 h. Normal animals spend equal time exploring each object during the acquisition phase, but spend more time exploring the newly introduced object versus familiar one during the retrieval. If object memory is impaired, during the retrieval phase the animal treats both objects as unfamiliar, and thus spends equal time exploring each object.

Episodic Memory These tests evaluate the animal’s ability not to merely discriminate between earlier encountered and novel objects, but to put these encounters in the context of the environment, by introducing the “What” happened “Where,” and in “Which” context (WWWhich), or “What” happened “Where,” and “When” (WWWhen) scenarios (Eacott and Norman, 2004; Inostroza et al., 2013; Kart-Teke et al., 2006). WWWhich version. The animal is successively exposed to different contexts (Eacott and Norman, 2004), as i­ llustrated in Fig. 13.1B. Context 1 consists of an open field with the mesh floor, and two objects with distinct shapes—a cube on the left, and a cylinder on the right (Fig. 13.1B, i; the left–right reference can be defined by placing a distinctly shaped sign in a fixed location, represented by the star). Context 2 consists of an open field with a smooth floor,

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with the cylinder on the left, and the cube on the right (Fig. 13.1B, ii; the cube and the cylinder are exact copies of the objects from the Context 1, but their locations are swapped). The animal is first exposed to Context 1, and immediately after that to Context 2, each exposure lasting 10 min. During the two exposures, the animal is expected to spend equal time exploring each of the objects. After the two exposures, the memory is tested with a series of delays between exposure 2 and the test ranging between 2 and 120 min. For this, the animal is placed in the open field with the mesh floor (i.e., the one from Context 1). However, it is now presented with two identical objects—the cubes on the left and the right. Both objects are familiar in shape, but the cube on the right is new in the context (compare object locations in Fig. 13.1B). A normal animal is expected to spend more time exploring the cube on the right, than the one on the left (Fig. 13.1B, iii). By varying the delay between exposure 2 and the test, the longevity of the episodic memory can be gauged. WWWhen-type tasks have a purpose similar to WWWhich-type assays; however, the “when” element of episodic memory has been proven problematic (Eacott and Norman, 2004) for developing tasks for rodents. Object recognition tests are relatively short lasting, a fact that makes them better suited for use in epileptic animals. However, as the tasks involve indifferent stimuli, even normal animals may show no novel object/context preference. Furthermore, the affinity toward the novel object/context may be diminished in the presence of neophobia. Discriminating between object memory and mood/anxiety impairments may be difficult.

Context Memory Contextual and cue fear conditioning are used to examine the animal’s ability for associative learning (Curzon et al., 2009a). The general principle is combining an otherwise neutral conditioned environment (i.e., the context), and/or a stimulus (i.e., the cue, such as a tone) with an aversive unconditioned stimulus (e.g., foot shock). Typical fear response in rodents consists of freezing; the presence and the length of freezing gauges the animal’s ability to learn to anticipate the unconditioned stimulus after the conditioned stimulus is presented. Conditioned and unconditioned stimuli may be temporally closely spaced (e.g., either presented simultaneously, or the unconditioned stimulus immediately follows conditioned stimulus). This design is referred to as delayed conditioning, and is used to examine the involvement of amygdala (Vazdarjanova and McGaugh, 1998). Alternatively, conditioned and unconditioned stimuli may be separated by some time (seconds to 1 min); this is called trace fear conditioning, and is used to infer hippocampal (particularly CA3) involvement (Curzon et al., 2009a).

The most commonly used experimental design is the 2-trial delay cued and contextual fear conditioning.

2-Trial Delay Cued and Contextual Fear Conditioning The test is performed in a chamber equipped with a grid floor and a sound source. On the first day, after several minutes of habituation, a 70–80 dB auditory stimulus is delivered for 15–30 s. During the last 2 s of the tone, a mild electroshock (0.5 mA) is administered, and it coterminates with the tone. After the shock, a 1–3 min interval precedes the next identical trial. After the second trial, the animal is returned to the home cage. On the second day, contextual fear is examined first. The animal is placed in the same environment that was used on the first day (i.e., same chamber, located in the same room), and is observed for 3 min with no tone presented; the presence of freezing at this time is the evidence of contextual fear. The animal is returned to the home cage for 30 min. Next, cued fear is examined. The animal is placed into a different context environment (i.e., different room with different lighting, odor, and conditioning chamber). After 2 min of habituation, the animal is exposed to the conditioned stimulus used on day 1. The presence of freezing behavior is an indicator of cued fear. For the traced cued and contextual fear conditioning, the setup is essentially the same, except that the conditioned stimulus is delayed by 5–60 s. Traced fear conditioning requires five trials. The quantification includes measuring the duration of freezing behavior. Shortening of freezing time indicated deficits in associative memory. Conversely, the increased duration of freezing may be an expression of generalized anxiety or phobia-like impairments (see Anxiety Disorders). Development of seizures shortly after the procedure on day 1 may interfere with memory formation, and complicate discerning between chronic and postictal contributing factors. The involvement of frontal lobes in associative memory (Barker and Warburton, 2008; Hales and Brewer, 2011) may be a further confounding factor: particularly, the presence of attention deficit may produce false positive result.

Social Transmission of Food Preference The test can be used to measure both contextual memory and communication ability, the latter being impaired in animals with autism-like abnormalities (Galef, 2003; Wrenn, 2004). Therefore, discerning between underlying causes may be complicated, and must be viewed in the context of the ­outcomes in other relevant tests (see Autism/­ Impaired Communication).

DEPRESSIVE DISORDERS Behavioral tests are summarized in Table 13.2.

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TABLE 13.2 Tests for Depression and Anxiety Test Description

Examined Symptom

Signs of Impairment

Forced swimming test (single session, or Porsolt)

Increased immobility

Tail suspension test (mouse only)

Inability to cope with stressful situation; despair; fatigue; indecisiveness

Saccharine/sucrose preference

Anhedonia

Lack of preference toward saccharin/sucrose versus tap water

Sexual behavior

Sexual dysfunction; anhedonia

Increased mount, intromission and ejaculation latencies; decreased mount, intromission and ejaculation frequencies

Sleep structure

Dyssomnia

Shortened REM latency, increased REM episode number and duration

DEX suppression; combined DEX/CRH

HPA axis dysregulation

Bunted response to DEX Exacerbated response to CRH

Neuroendocrine response to immobilization stress

Exacerbated increase of plasma corticosterone

Elevated plus maze

Generalized anxiety

Increased presence in closed arms/reduced presence in open arms

Open field

Generalized anxiety

Increased presence in peripheral versus central areas

Three-chamber test

Social anxiety

Avoidance of a conspecific

Contextual and cued fear conditioning

Fear

Increased freezing upon presentation of a conditioned context and/or stimulus

DPAG stimulation-induced behavioral responses

Panic disorder

Decreased threshold for inducing panic-like behaviors

Despair, Hopelessness, Fatigue Forced Swimming Test Under conditions of FST, an inescapable stressful environment is created by placing an animal in a cylindrical tank filled with water, with no escape options, typically for 5 min (Castagne et al., 2009; Mazarati et al., 2010;

­ verstreet, 1993, 2012; Petit-Demouliere et al., 2005). O ­Animals display several behavioral patterns (Fig. 13.2A) most notably active swimming (i.e., swimming along the walls, climbing, diving) that is interpreted as effective coping (i.e., devising an escape plan). Intermittently, the a­ nimals show periods of immobility (i.e., passive floating, with movements limited to maintaining the head

FIGURE 13.2  FST and combined DEX/CRH tests. (A) FST. Sample snapshots taken from prerecorded video during FST in a Wistar rat. Time after the start of the test is indicated on each image. Examples of active swimming are presented at 1 min 34 s and 1 min 37 s. Examples of immobility are presented at 2 min 58 s and 3 min 04 s. (B) Combined DEX/CRH test. Plasma corticosterone levels in naïve rats (Control) and chronic epileptic rats (Post-SE; 3 months after LiCl and Pilocarpine SE). “Before SE/Saline” refers to the plasma corticosterone level before SE. The rest of the time points describe DEX/CRH test. Plasma corticosterone levels are shown before DEX injection, 6 h after DEX injection, 30 min and 60 min after CRH ­injection. Data are presented as mean ± SEM. *P < 0.05 versus “before SE/saline”; #P < 0.01 versus “before DEX/CRH”; ††P < 0.01 and †††P< 0.001 versus “DEX-6 h.” (Part A: Reprinted from Mazarati, A.M., Pineda, E., Shin, D., Tio, D., Taylor, A.N., Sankar, R., 2010. Comorbidity between epilepsy and depression: role of hippocampal interleukin-1beta. Neurobiol. Dis. 37, 461–467, with permission from Elsevier; Part B: Reprinted from Mazarati, A.M., Shin, D., Kwon, Y.S., Bragin, A., Pineda, E., Tio, D., Taylor, A.N., Sankar, R., 2009. Elevated plasma corticosterone level and depressive behavior in ­experimental t­emporal lobe epilepsy. Neurobiol. Dis. 34, 457–461, with permission from Elsevier.)

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above ­water). In normal animals, periods of immobility generally account for less than one-third of total swimming time, and reflect resting and/or deciding on a new escape strategy. In validated depression models, animals spend significantly more time immobile. The increment in immobility is regarded as a failure to cope with stress, and thus may reflect a state of despair, hopelessness, fatigue, or indecisiveness. The severity of these impairments is generally expressed as total duration of immobility, although discriminating among various mentioned states is hardly possible. Climbing behavior has a strong noradrenergic component, whereas other active behaviors (i.e., swimming along the walls and diving) are driven mostly by serotonergic transmission (Detke et al., 1995) (although dopamine has been implicated in regulating both behaviors; Espejo and Minano, 1999; Perona et al., 2008). Therefore, calculating at the expense of which active type of behavior the increase of immobility occurs may help in determining which monoaminergic deficits are predominant in the test animal. FST used for gauging depressive behavior should be discerned from FST as a model of depression. Classical Porsolt test includes two swimming sessions, presented within several days (Porsolt et al., 1979; Petit-Demouliere et al., 2005). The first session is depressogenic in an inherently normal animal. The second session is testing that evaluates the animal’s behavior. With regard to epilepsy, either a single-session, or a two-session FST can be used, depending on goals of the experiment. If the goal is to establish whether epilepsy itself creates depressive state, then single FST is appropriate. At the same time, a scenario is possible when epilepsy proper does not lead to depression, but instead primes the animal to it. In such cases, the Porsolt test may be useful, as subjecting the animal to the first swimming trial may exacerbate the severity of depressive behavior during the second session. For the FST, intact balance and coordination are critical. Damage to vestibular structures may lead to animals spending more time actively swimming. Such swimming, however, has no escape purpose (e.g., animals will be intensively treading water in the middle of the tank). It has also been suggested that such uncued active swimming may be an expression of hyperimpulsivity, and thus may be observed in animals with ADHD-like abnormalities (Pineda et al., 2014). This can be corroborated using relevant tests (see Attention Deficit/Hyperactivity Disorder). In mice, the tail suspension test is frequently used in lieu of FST, with the same premise (Castagne et al., 2009). When hanged by the tail, the mouse exhibits two behaviors—struggling, and immobility—that correlate with active swimming and immobility in the FST, respectively. Cumulative duration of the immobility provides a measure of depressive behavior.

Anhedonia Taste Preference The taste preference test relies on the inherent affinity of rodents toward sweets; the lack of preference toward sweet drink versus regular water is inferred as anhedonic state (Harkin et al., 2002; Overstreet, 2012). For the test, animals are housed individually in their home cages equipped with two bottles. During the first day of the test (habituation), each bottle contains regular water. After 24 h, the remaining volumes are measured; the volumes should be similar between the two bottles (otherwise suggests a presence of a bias that should be corrected). On the second day, the content of one of the bottles is replaced with a sweet solution—commonly either 1% sucrose, or 0.1% saccharine. After 24 h, the remaining volume in each bottle is measured. Normal animals consume more sweet solution than regular water whereas, in validated models of depression, animals consume equal volumes of each. In order to exclude the bias, the test can be extended to the third day, when the bottles are refilled, and the positions are switched. As the test relies on intact taste, if the outcome of the test does reveal the lack of taste preference, a control experiment should be performed in order to exclude deficit in taste sensation. Rodents avoid consuming bitter substances, for example, quinine solution. The taste aversion test is performed similarly to the saccharine/sucrose preference test, except that one of the bottles contains 10 M quinine. Animals with preserved taste would consume little, if any, quinine solution.

Sexual Behavior In rodents, impaired sexual behavior may be a result of either diminished sexual drive, or of anhedonia. The evaluation of sexual behavior is commonly performed in males vis-à-vis ovariectomized, estradiol/progesterone-treated, age-matched females. Ovariectomy and hormone treatment are performed in order to standardize estrus cycle, as the latter plays key role in determining male mating behavior (Gronli et al., 2005). Preparation of female partners. Female animals are ovariectomized at least 2 weeks before the test. Forty-eight hours before mating, females are injected with estradiol (50  µg/kg); 6 h before mating, animals are injected with 0.1 mg/kg progesterone. Preparation and selection of test male animals. Males should be sexually inexperienced at the beginning of the experiment. Prior to the test, the female rat is introduced into the home cage for 30 min, and the male’s rat behavior is recorded. Mating is always carried out during the dark phase of 12-h light/dark cycle. The session is repeated during 3 consecutive days. The males are used for further experiment only if they have a total of three ejaculations

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during the training. Otherwise, the animals are identified as noncopulators and are excluded. Test proper. The female rat is introduced into the home cage for 30 min, and the male’s behavior is video recorded. The following behaviors are recorded: (1) mount latency: time elapsed between introducing the female and the first mounting trial without intromission; (2) mount frequency; (3) intromission latency: time elapsed between i­ntroducing the female rat into the male cage and the first ­intromission; (4) intromission frequency; (5) ejaculation latency: time elapsed between the first penetration and ejaculation; (6) ejaculation frequency. Depression is characterized by increased latencies and decreased frequencies, for any of the parameters, as compared with normal subjects.

Dyssomnia Depression is characterized by perturbations in the sleep structure, notably by the shortening of latency to the Rapid Eye Movement (REM) sleep, increased number of REM sleep episodes, and of the REM sleep duration (Tobler et al., 1997; Popa et al., 2008).

Analysis of sleep structure For comprehensive sleep analysis, it is recommended to simultaneously record EEG, electrooculogram (EOG), and EMG from neck muscles. At least 4 consecutive days of video/EEG recording is recommended, with animals residing in home cages at 12-h dark/light cycle and free access to water and food. Waking state is characterized by high EMG and low EEG amplitude, and high theta activity concomitant with highest EMG values. Non-REM sleep is characterized by low EMG amplitude, high EEG amplitude, high delta activity, absence of EOG activity. REM sleep is characterized by low EMG and low EEG amplitude, high theta activity, high EOG activity. Spontaneous seizures developing during sleep are disruptive and may affect sleep structure independently of chronic epileptic state.

Neuroendocrine Correlates Dexamethasone (DEX) Suppression and Combined DEX/CRH Tests The test involves several venous blood collections (typically from the tail vein, although femoral vein catheterization may be required, particularly in mice), and subsequent assay of corticosterone plasma or serum levels (Hatzinger et al., 1996; Mazarati et al., 2009). At least 10 µL of plasma is required. The first sample is collected in order to detect baseline corticosterone level. At the end of the collection, DEX is injected intravenously at 30 µg/kg. The second sample is drawn 4–8 h after DEX injection. For the DEX suppression test, this is the last collection. If the combined

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DEX/CRH test is conducted, after collecting the second sample, the animal is injected intravenously with CRH at 50 ng/kg, and two subsequent samples are collected 30 and 60 min after CRH injection. Combined DEX/CRH test has been suggested to be more accurate for detecting HPA axis dysregulation; however, given its longer duration and thus a higher chance of seizure occurrence, its feasibility may be limited. Corticosterone content in plasma or serum samples is detected by means of radioimmunoassay. Normal response to DEX injection consists of approximately threefold decrease of plasma corticosterone level. The dysregulation of the HPA axis is characterized by the blunted response to DEX. Normal response to CRH consists of two- to fivefold increase of plasma corticosterone level at 30 min, and by its return to the pre-CRH level at 60 min. If the HPA axis is hyperactive, the increase is significantly exacerbated and is still present at the 60 min time point (Fig. 13.2B).

Neuroendocrine Response to the Immobilization Stress Conceptually, the test is analogous to the CRH challenge part of second part of the combined DEX/CRH test. Here, the spike in the corticosterone level occurs after the animal is restrained for 30 min, with three blood collections, one before and two after the restraint (at 30 and 60 min). The dysregulation of the HPA axis is characterized by the exacerbated increase of plasma corticosterone (Jones et al., 2013; Pitman et al., 1988). Recurring seizures taking place during both DEX/CRH test and the immobilization stress may produce additional increase in blood corticosterone, which may last for several hours.

ANXIETY DISORDERS Generalized Anxiety Disorder Elevated Plus-Maze Behavior in the EPM is determined by the balance ­between the rodent’s innate aversion to open spaces, and their natural curiosity (Bailey and Crawley, 2009; Walf and Frye, 2007). A plus-shaped apparatus consists of two open arms, crossed at the right angle by two closed arms. ­Additional sense of insecurity is created by placing the apparatus above the floor level. The animal is placed at the crossing point (which is exposed) and is allowed to explore the apparatus for 5–10 min. The number of entries, and total time spent in closed versus open arms, is calculated. Rodents always prefer closed arms, but their presence in open arms is noticeable (due to curiosity). The increased time spent in closed arms/decreased presence in open arms is treated as an indicator of generalized anxiety.

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While positive EPM test has been reported in rodent models of epilepsy (Duarte et al., 2013; Muller et al., 2009), several studies found that epileptic animals spend more time in open arms than their normal counterparts—which, at the surface, may be interpreted as reduced anxiety (Detour et al., 2005; Faure et al., 2013; Jones et al., 2009). It is hard to conclude whether seizures genuinely produce anxiolytic effects, or whether the animals’ behavior is affected in some other ways that render EPM inappropriate for examining anxiety. For example, it has been shown that in rats with pilocarpine SE-induced epilepsy, the increased presence in open arms of EPM positively correlates with the severity of ADHD-like impulsivity (Pineda et al., 2014). It is thus possible that what appears to be a decreased anxiety in fact represents a nonspecific manifestation of impulsivity.

Open Field The rationale behind the open field test is the same as behind the EPM test. In contrast to the EPM, the animals are offered more degrees of freedom, in the open field, thus displaying more complex behaviors. On the one hand, this allows for more detailed analysis of animals’ behavior but, on the other hand, may make the interpretation even more complicated (Bailey and Crawley, 2009). Open field is a large square area surrounded by walls, and divided in 16–25 squares by painted lines. The animal is placed in the center, and is allowed to explore the field for 5–10 min. Several parameters are analyzed, including the number of entries into, and time spent in, peripheral areas (i.e., squares adjacent to the walls) versus central areas (i.e., squares nonadjacent to the walls), and the number of crossed squares. Rodents always prefer peripheral areas over central ones. The main indicator of anxiety is a further increase of time spent on the periphery, up to full avoidance of central areas.

Social Anxiety Disorder All tests described under Autism Spectrum Disorder/­ impairments in social interaction can be used to evaluate social anxiety in rodents. In the context of anxiety, avoidance of conspecifics is interpreted as social anxiety. Discerning between purely impaired sociability and social anxiety can be complicated. For example, social anxiety is a more likely diagnosis, if during the sociability phase of the threechamber test, the test animal spends more time away from the conspecific (i.e., either in the central chamber, or in the one with the object), rather than divides the time between the conspecific and the object equally. Similarly, during the social novelty phase, an anxious animal would be avoiding both familiar and novel conspecific by spending more time in the center chamber, or would prefer familiar conspecific over the novel one. The absence of other core symptoms of autism would suggest anxiety rather than ASD-like impairments.

Fear Several fear conditioning tests (such as contextual and cued fear conditioning) may be used for studying phobia-like behaviors. As the tests rely heavily on contextual memory (as described earlier), interpretation of outcomes of these tests in the context of anxiety may be challenging. Generally, the prolonged freezing suggests the presence of phobia-like impairments (as opposed to shortening of freezing that points toward deficit in associative memory). A combination of associative memory deficits with phobia complicates the data interpretation significantly. Fear conditioning is described under learning and memory impairments.

Panic Disorders Panic behavior is mediated by dorsal periaqueductal grey matter (DPAG) (Jenck et al., 1995; Siqueira et al., 2010). In rodents, panic-like responses can be elicited by titrated electrical stimulation applied to DPAG; quantification is based on determining stimulation thresholds for inducing typical behavioral reactions (Quintino-dos-Santos et al., 2014a,b). Animals are implanted with chronic stimulating electrode into DPAG. Electrical stimulation is performed in free-moving animals. Typical parameters are 30 s trains, 60 Hz, starting with 5 µA, with 5 µA increments, applied 5 min apart. Behavior is analyzed during the stimulations as follows: (1) exophthalmos: eyeball protrusion; (2) ­immobility: behavioral arrest accompanied by the increase of muscle tone in the neck and limbs; (3) trotting: fast ­locomotion with out-of-phase stance and swing movements of contralateral limbs, and the elevation of trunk and tail; (4) galloping: running alternating stance and swing movements of anterior and posterior limb pairs; (5) jumping: upward leaps; (6) defecation and micturition: ejection of feces and urine. Minimal current required to induce each of the described behavioral reactions is determined.

AUTISM SPECTRUM DISORDERS ASD is characterized by three core symptoms: impaired social interaction, impaired communication, and repetitive behaviors (American Psychiatric Association, 2013). ­Behavioral tests are summarized in Table 13.3.

Impairments in Social Interaction Three-Chamber Test The test relies on the fact that rodents readily engage with conspecifics, particularly with those that they have not encountered before (Crawley, 2007). The test is performed in an apparatus divided in three horizontally arranged equally-sized chambers, connected at

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TABLE 13.3 Tests for Autism Test Description

Examined Symptom

Signs of Impairment

Three-chamber: sociability

Impaired social interaction

Lack of preference toward a conspecific versus an unanimated object

Three-chamber: social novelty Social transmission of food preference

Lack of preference toward an unfamiliar conspecific versus familiar conspecific Impaired communication

Lack of preference toward a conspecific-cued food

Ultrasonic vocalizations in the resident-intruder test (adults)

Reduced number of ultrasonic calls in the presence of a conspecific

Ultrasonic calls upon maternal separation (pups)

Reduced number or altered structure of ultrasonic calls upon separation from the dam

Marble burying

Compulsive behavior

Increased number of buried marbles

Self-grooming

Ritualistic and stereotype behaviors

Prolonged duration of self-grooming

Reversal learning in T-maze (simple and probabilistic)

Behavioral rigidity/insistence on sameness

Lack of ability to switch to a new location of food reinforcer

Morris water maze

the bottom level with openings large enough for the animal to travel between the chambers. Each of the terminal chambers contains a cylindrical wired enclosure, with the footprint occupying 10%–15% of the chamber surface (Fig. 13.3A–B). Prior to the test, the animals are housed individually over several days, so as to stimulate the interest toward conspecifics upon encounters. The test runs in three consecutive 10-min sessions. The first session is habituation, when both wired en-

Delayed ability to learn new location of the platform

closures are empty. The second session tests animal’s ­sociability. For this purpose, an unfamiliar strain-, age-, and sex-matched animal (conspecific 1) is placed inside one of the wired enclosures, and an unfamiliar indifferent object (sized approximately as the conspecific) inside the other enclosure (Fig. 13.3A). The third session examines perception of social novelty, for which the object is ­replaced with another unfamiliar conspecific (conspecific 2, Fig. 13.3B).

FIGURE 13.3  Tests for social interaction and communication. (A–B) Three-Chamber Test. (A) During the sociability phase, the test animal (outside the wired enclosure) interacts with the conspecific (right) and the object (left). (B) During the social novelty phase, the object is replaced by a new conspecific (left), while the old conspecific is the same as in (A). (C) An example of ultrasonic vocalization in the resident-intruder test. Test animals are adult males residing in home cages. In the top portion, the resident in a normal B6 mouse; in the bottom portion the resident is a mouse of BTBR strain. In both cases, the intruder is the same female B6 mouse. Ultrasonic calls are emitted at 55–85 kHz frequency. Samples of 5 s are shown. Ultrasonic calls are indicated dots. (D) Social transmission of food preference. For the olfactory cue acquisition, the demonstrator (marked as “D”) first consumes cocoa-flavored food. Next, the demonstrator transmits the cue to the test animal (observer, “O”). The observer is then given the choice between the cued (cocoa-flavored) and noncued (cinnamon-flavored). If the interaction between the demonstrator and the observer was adequate, the observer preferentially consumes the cued food.

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During the sociability phase, normal animals engage more with the conspecific than with the object (and/or spend more time in the chamber where the conspecific is located, but see discussion later). During the social novelty phase, normal animals engage more with the novel conspecific than with the familiar one. Impaired sociability manifests itself as the lack of preference toward the conspecific during the second session, and/or to the novel conspecific during the third session of the test. This lack of preference is evident in that the test animal engages equally with the conspecific and the object during the sociability phase, and with the familiar and the novel conspecifics during the social novelty phase. The animal’s behavior can be analyzed in several ways. Automated methods often calculate the presence of the test animal in each of the chambers, with the assumption that normal sociability is characterized by the bias toward the chamber with the conspecific during the sociability phase, and with the novel conspecific during the social novelty phase. However, such method may not be sensitive enough for revealing impairments in social behavior, even in validated autism models (Pineda et al., 2013; Hagen et al., 2015). Moreover, autism-like abnormalities may include displacement behavior, whereby the animal’s presence in the chamber with the conspecific/novel conspecific is increased, but the animal shows ritualistic behavior (typically self-grooming) (Hagen et al., 2015). A more sensitive way to analyze the behavior appears in quantifying direct engagement attempts (i.e., sniffing and attempts to reach for the conspecific and the object) (Crawley, 2007). As rodents rely heavily on olfaction, anosmia may produce false results. Intact short-term object memory may be important for the social novelty phase. These considerations may be of particular importance when dealing with epileptic animals, when there is a possibility that either a precipitating insult or recurrent seizures may affect olfaction and short-term memory. Correction assays, such as buried food test, and novel object recognition test, may help in revealing olfaction and memory deficits respectively. Social anxiety even not related to autism may manifest itself as an active avoidance of a conspecific altogether. A more complicated scenario may ensue if the strength of sameness (i.e., resistance to change that, in itself, is a symptom of autism) overrides the strength of social disengagement. For example, an animal with strongly expressed behavioral rigidity may simply stick to a chamber that it randomly chooses during the habituation phase throughout the test, notwithstanding the content of the enclosure during the subsequent sessions. The outcome of a respective test may provide some clues (see later).

Reciprocal Interactions The three-chamber test is unidirectional. Therefore, the analysis of reciprocal social interaction is often used to ­supplement the three-chamber test, whereby the test animal

and the conspecific are not separated. The test is normally performed in an environment that is unfamiliar to both animals (e.g., novel cage or open field), and lasts 10 min. The interaction of the test animal with a single stranger, and with the group of 3–5 strangers, is analyzed by q­ uantifying such behaviors as nose-to-nose sniffing, ano-genital sniffing, following, allogrooming, and fighting. Poor first four behaviors point toward impaired sociability, whereas increased fighting may suggest aggression that may (or may not) be associated with autism.

Impairments in Social Communication Ultrasonic Vocalizations Rodents communicate by means of ultrasonic vocalizations (USV) and, in validated autism models, USV are frequently impaired. The impairments vary significantly, from simple reduction in the number of ultrasonic calls in the presence of conspecifics, to complex changes in modality, tonality, structure, duration, etc. (Crawley, 2007; Scattoni et al., 2008, 2011; Malkova et al., 2012). USV are induced using different communication scenarios. In rodent pups, due to overall poor behavior, the analysis of USV is the only objective way to measure communication abilities. Normal pups emit ultrasonic calls of defined frequency and patterns, upon their separation from the dam. In autism models, upon maternal separation, the number of calls emitted by the pups may be decreased either overall, or selectively (e.g., reduction of complex, upward, chevron, and frequency step calls), while the total number of USV may remain unaltered (Scattoni et al., 2008, 2011; Malkova et al., 2012). USV analysis can be used for investigating autism-like impairments in adult rodents, normally using a resident–intruder scenario. The resident is the test animal residing in its home cage. The intruder is an unfamiliar age-matched animal (that may be of either the same or the opposite sex, depending on the study goals), is introduced into the resident’s cage for 3–5 min. During the resident–intruder interaction, the latter is typically silent, while USV are emitted by the resident. Autism-like impairments are characterized by the reduced number of USV (Fig. 13.3C).

Social transmission of food preference Nonauditory interaction can be measured using Social transmission of food preference (STFP). The test is based on the preference of rodents toward food bearing a certain olfactory signature that is deemed safe, as opposed to food with an unknown olfactory signature. The perception of safety comes from an interaction with another animal that has previously consumed this specific type of food (PosadasAndrews and Roper, 1983; Wrenn, 2004). The test involves a test animal (observer), and a conspecific (demonstrator). Prior to the test, the demonstrator is limited to consuming

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food with one of the two distinct flavors (typically cinnamon and cocoa). The demonstrator is then allowed to interact with the observer, after which the observer is presented with the choice between the cued and noncued food. For example, if the demonstrator consumes cocoa-flavored food, cocoa serves as a cue during the test, whereas cinnamon serves as a noncued food. If the interaction between the demonstrator and the observer was sufficient, the observer picks the olfactory cue, and subsequently preferentially consumes the cued food. If the interaction was lacking, then during the test it would consume equal amounts of cued and noncued food (Fig. 13.3D). The strength of social interaction can be expressed as a percent of cued food of a total food (i.e., cued + uncued) consumed by the observer. Similarly to the three-chamber test, intact olfaction is required for the test to be valid. Anhedonia may lead to overall low food consumption, and thus invalidate the test result. Discerning between mere lack of interest in conspecific and its active avoidance, due to social anxiety, may be complicated.

Repetitive Behaviors The term “repetitive behaviors” pertains to various behavioral deficits, including ritualistic, stereotype, compulsive behaviors, and behavioral rigidity (the latter is also described as insistence on sameness, or resistance to change) (American Psychiatric Association, 2013).

Self-Grooming The most common way to analyze behavioral rituals is through the quantification of self-grooming, including the number and the duration of grooming episodes during a set period of time (typically 10 min). As a part of normal behavioral repertoire, grooming is episodic, but it becomes overrepresented in autism models, and is interpreted as an analog of either stereotypy, or a ritual reported in autism patients. Grooming can be observed under different conditions. Most commonly, the animal is placed individually inside a restricted enclosure, but the observations can be done in the home cage (if the animal is housed individually), or even during the three-chamber test (Crawley, 2007; Malkova et al., 2012; Hagen et al., 2015). Depending on the design, the test allows analyzing behavior in isolation or in the context of the environment. During the three-chamber test, animals with autism-like impairments may spend more time grooming in the presence of a conspecific/novel conspecific, fact that can be interpreted as a displacement behavior (Hagen et al., 2015).

Object Burying Obsessive behavior can be quantified using the object-burying test (Thomas et al., 2009; Malkova et al., 2012). ­Burying

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unfamiliar uniform objects under the layer of bedding is interpreted as an analog of object rearrangements. The animal is placed in an unfamiliar cage for 10 min, where 20 uniform shaped odorless objects (typically small marbles) are evenly distributed on the top of the bedding. Normal animals bury around 50% of the objects during the test, while in validated autism models animals bury significantly more.

Behavioral Rigidity Behavioral rigidity, that is, resistance to adapt behavior to changes in the environment, is common among patients with autism. It is generally analyzed using a variety of reversal learning tests (Moy et al., 2007; Amodeo et al., 2012).

Reversal Learning in the T-Maze The apparatus is a T-shaped maze wide enough for the animal to travel along the arms, but narrow enough to prevent the animal from wandering within a particular arm (Fig. 13.4). One of the arms is designated as “correct.” At the end of this arm, a food pellet is placed. The opposite is the “incorrect” arm (Fig. 13.4A). The test consists of three consecutively run phases, lasts for 12–14 days, and the animal is under food restriction throughout the test. During the habituation phase, the food is not presented, and the animal is allowed to explore the maze freely. During the acquisition phase, the food is placed in the correct arm, the animal is repeatedly placed into the base of maze, and each time is allowed to explore the apparatus until it locates the food and consumes it. After several dozens trials, the animal learns which arm contains the food pellet, and the acquisition ­culminates in the animal consistently walking into the “correct” arm. A criterion to success is six consecutive visits to the “correct” arm. For the subsequent reversal learning phase, the location of the food sample is switched to the opposite arm, and the procedure is repeated. Normal animals learn fairly quickly the new location of the food. Behavioral flexibility is quantified by calculating the number of trials needed to learn the new food location (i.e., six consecutive visits to the “correct” arm; normally, this number is similar to the number of trials required to accomplish the acquisition phase). Delayed learning during the reversal phase, or complete inability to switch to the new location, is regarded as behavioral rigidity. In some validated autism models, behavioral rigidity in the simple reversal learning test is not detectable. For example, BTBR mice that present with several autism-like impairments perform well in the simple reversal learning test (Amodeo et al., 2012). Therefore, a modified version of the test has been developed. Instead of placing the food consistently into one of the arms, it is placed in the “correct” arm in 8 out of 10 trials, and in the remaining 2 out of 10 trials the food is present in the “incorrect” arm. For the

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FIGURE 13.4  Reversal learning in the T-maze. (A) Simple reversal learning. Food pellets placed in the right arm during consecutive acquisition trials, as denoted by numbered circles. Once the animal is trained, the food pellet location is switched to the left arm for the reversal-learning phase. The number of trials required for the animal to visit the new “correct” arm is calculated. (B) Probabilistic reversal learning. During acquisition trials 3 and 8, the food pellet is placed in the right arm, while during all other trials in the left (“correct”) arm. The animal is expected to learn the chance for finding the food on the left is higher than on the right. Upon accomplishing acquisition phase, the food locations are switched for the reversal learning.

reversal learning, the 8:2 ratio is switched (Fig. 13.4B). In such probabilistic version, the animal has to learn the arm that has a better chance of containing the food (Amodeo et al., 2012). This version of the test allows revealing behavioral rigidity in animals that behave normally in the simple test, including BTBR mice. As for other tests, several requirements should be met in order for the test to be valid. Those include preserved spatial and context memory, and absence of anhedonia. As behavioral rigidity is also a symptom of schizophrenia, discerning between autism-like and schizophrenia-like abnormalities should be performed in the context of behavioral perturbations observed in relevant assays. Behavioral rigidity can be examined as a part of the Morris Water Maze paradigm. This can be useful for combining studies of behavioral rigidity with examining spatial learning and memory. For this, upon completing probe trials, the platform is moved to a new quadrant (for details, see Spatial Cognition and Memory/Morris Water Maze).

ATTENTION DEFICIT/HYPERACTIVITY DISORDER (ADHD) Attention deficit and hyperactivity can be examined ­simultaneously, as parts of the same behavioral test. There are several tests for ADHD, all representing operant tasks with positive reinforcement. The animal is trained to react in a certain way when presented with a sensory stimulus; if

a response fits the criteria, the animal is rewarded, and vice versa. Based on the number of errors, conclusions on the presence of attention deficit and/or hyperimpulsivity can be drawn.

The 5-Choice Serial Reaction Time Task The test is performed in an operant conditioning box in which five holes with infrared beam detectors are located on one wall, and a food magazine is located on the opposite wall (Robbins, 2002; Sanchez-Roige et al., 2012). Above each hole, there is a light. Prior to and during the test, the animal is on food restriction, with the target body weight at 85% of the baseline. Prior to training, the animal is allowed to habituate in the operant chamber, and to learn that the food magazine dispenses food pellets upon nosepoking a hole. The training consists in sequentially switching each of the lights on and off, and the animal learning that food is only dispensed if it pokes the hole under the correspondent light, and only after the light is switched off. Poking an ­incorrect hole points toward lack of attention; poking a ­correct hole, but prematurely, points toward ­hyperimpulsivity. Several types of errors are measured: (1) attention errors: visits to the wrong hole. Increased number of visits to a wrong whole is an evidence of attention deficit; (2) omissions: failures to respond within 5 s after the light is off. Increased number of omissions also suggests lack of attention; (3) premature responses, when the animal reacts

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before the stimulus sequence is completed. Increased number of premature responses is an evidence of hyperimpulsivity (Robbins, 2002; Sanchez-Roige et al., 2012). The duration of the intertrial interval and the duration of the stimulus presentation can vary, depending on the goals. Shortening intervals between stimuli increase attention errors. Increasing intertrial intervals increase a chance of premature responses.

Lateralized Reaction-Time Task The operant chamber, as well as the principle, is similar to the 5-choice serial reaction time task (5-CSRT). In lateralized reaction-time task (LRTT), the animals engage in a variable-duration fixation response, while waiting for the delivery of a visual target (light) in the left or right visual fields. During this fixation response, the animals must divide and orient their attention, monitoring both locations for the stimulus delivery; because the temporal onset and duration and spatial localization of the target are unknown to the rats, the subjects will “miss” the target if they attend to only one location and/or fail to sustain their attention. Dependent measures include: (1) discriminative response accuracy (i.e., correct responses/[correct + incorrect responses]): measure of attention; (2) omission rate (as a percent of total trials): measure of attention; (3) total anticipatory responses: measure of impulsivity; (4) mean initiation latency/trial (i.e., the average interval between illumination of the center nose poke aperture and the initiation of the observing response): measure of impulsivity (Jentsch, 2005; Jentsch et al., 2009). Both assays take several weeks to complete. Therefore, recurrent seizures are likely to interfere with the training and performance, and thus significantly affect the outcomes of the tests (Faure et al., 2014; Pineda et al., 2014).

SCHIZOPHRENIA Impairments in Sensory Gating Sensory gating is the ability of the central nervous system to adapt to sensory stimuli upon their repeated presentation. It is commonly impaired in schizophrenia patients, and may relate to the inability to concentrate, and to the overload of attended stimuli (Cromwell et al., 2008). Several variants of the test have been adopted in rodents (Curzon et al., 2009b).

Standard Startle with Startle Habituation The test is performed in a startle box, enclosed in the sound-attenuating chamber. The chamber is equipped with a loudspeaker that delivers white noise and sounds of set ­intensities. The startle box is equipped with a motion ­sensor. The test begins with habituation, with the animal placed in the startle box, and exposed to background white noise (typically 65 dB) for 5 min. Afterward, the animal is e­ xposed

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to four sound bursts of 120 dB, 40 ms, so as to allow for the establishment of a stable baseline. Following this, acoustic startle trials are initiated. For simple assessment of acoustic startle responses, two or three stimulus intensities are used (90 and 105 dB, or 90, 105, and 120 dB). The stimuli are 40 ms long, and are presented in a quasirandom order, so that an equal number of presentations of each stimulus intensity are included in each half of the session, and no single intensity is presented more than twice in succession. The interval between stimuli varies within 5–30 s, so that the animal cannot anticipate the stimulus. The magnitude of the startle response is expressed, as the amplitude of the animal’s movement is triggered by the sound (the higher the amplitude, the stronger the startle). Normal sensory gating is characterized by progressive decrease of movement amplitudes upon repeated presentation of auditory stimuli; conversely, in animals with impaired sensory gating, movement amplitude does not diminish with time (Curzon et al., 2009b).

Prepulse Inhibition In this version of the test, the attenuation produced by a low intensity stimulus presented just before the startle stimulus is assessed. Following the 5 min habituation in the startle box with white noise, four successive trials of 40 ms noise bursts at 120 dB are presented. Subjects are then exposed to five different types of acoustic stimuli in a randomized order: pulse alone (120 dB noise for 40 ms), no stimulus (no stimulus is presented), and three separate prepulse + pulse combinations, with prepulse set at three sound levels of 70, 75, and 80 dB for 20 ms, followed by a 40 ms pulse at 120 dB. The prepulse-pulse interval is 100 ms. A total of 12 trials under each acoustic stimulus condition are performed, with intertrial intervals of 5–25 s (Curzon et al., 2009b). Under conditions of normal sensory gating, presentation of prepulse inhibits startle response to pulse stimulus; if ­sensory gating is impaired, prepulse stimulus fails to inhibit pulse-induced response.

Cognitive Rigidity Cognitive rigidity is typical, but not unique for schizophrenia (e.g., it is reported in autism patients, as discussed earlier). Reversal learning in T-maze, described under Autism, can be used. In the context of schizophrenia, more elaborate operant tasks have been developed.

Visual Discrimination Learning The animal is first trained to discriminate between two cues in exchange for food reward. Then, the cues are reversed, and the animal is expected to learn that a previously nonrewarded cue becomes rewarded, and vice versa. In a most common version, the animals are presented with two touch

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screens with two different images. Poking the nose in image A is rewarded by a food pellet dispersed from the magazine. Touching image B is not rewarded. Once the animal is trained in the discrimination task, the cues are reversed vis-à-vis the reward. Normal animals learn to switch to a rewarding image quickly, while animals with cognitive rigidity fail to adapt (Horner et al., 2013). The number of perseveration errors, defined as the inability to switch to the new rule, is used to measure cognitive rigidity.

Attentional Set-Shifting Attentional set-shifting is different from reversal learning in that the type of rewarded cue changes. For example, during training, visual cue is rewarded, while spatial cue (e.g., pressing lever) is irrelevant. Upon the completion of training, the cues are reversed. Perseveration errors are used to measure cognitive rigidity (Bissonette and ­Powell,  2012). Like other operant tasks, the described tests are long lasting, and interference of recurring seizures with animals’ training and performance should be factored in.

Psychostimulant-Induced Locomotion These tests are based in the dysfunction of dopaminergic neurotransmission in schizophrenia patients, and manifested in rodents as exacerbated behavioral responses to dopaminergic drugs. Apomorhine, a D1/D2 receptor agonist (0.75 mg/kg) induces climbing behavior. The latter is assessed by placing the animal into a cylindrical enclosure with walls constructed of smooth vertical metal bars. Climbing behavior is scored using an ordinal scale (0, all four paws on the floor; 1, gripping vertical bars with forepaws; 2, gripping vertical bars with four paws), and is exacerbated under conditions of dopaminergic hyperactivity. Amphetamine, an inhibitor of dopamine transporter (1.5 mg/kg), increases general locomotion that can be assessed in the open field by counting the number of crossed squares; dopaminergic dysfunction is evident as the increased number of squares crossed.

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