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. Author manuscript; available in PMC: 2015 Mar 18.
Published in final edited form as: Epilepsia. 2011 Sep 20;52(12):2293–2303. doi: 10.1111/j.1528-1167.2011.03267.x

Altered behavior in experimental cortical dysplasia

Fu-Wen Zhou *,†, Asha Rani †,‡, Hildabelis Martinez-Diaz *,†, Thomas C Foster †,‡, Steven N Roper *,†
PMCID: PMC4364520  NIHMSID: NIHMS657902  PMID: 21933180

Summary

Purpose

Developmental delay and cognitive impairment are common comorbidities in people with epilepsy associated with malformations of cortical development (MCDs). We studied cognition and behavior in an animal model of diffuse cortical dysplasia (CD), in utero irradiation, using a battery of behavioral tests for neuromuscular and cognitive function.

Methods

Fetal rats were exposed to 2.25 Gy external radiation on embryonic day 17 (E17). At 1 month of age they were tested using an open field task, a grip strength task, a grid walk task, inhibitory avoidance, an object recognition task, and the Morris water maze task.

Key Findings

Rats with CD showed reduced nonlocomotor activity in the open field task and impaired motor coordination for grid walking but normal grip strength. They showed a reduced tendency to recognize novel objects and reduced retention in an inhibitory avoidance task. Water maze testing showed that learning and memory were impaired in irradiated rats for both cue discrimination and spatially oriented tasks. These results demonstrate significant deficits in cortex-and hippocampus-dependent cognitive functions associated with the diffuse abnormalities of cortical and hippocampal development that have been documented in this model.

Significance

This study documents multimodal cognitive deficits associated with CD and can serve as the foundation for future investigations into the mechanisms of and possible therapeutic interventions for this problem.

Keywords: Comorbidity, Memory, Cognition

Comorbidities associated with epilepsy are common and may have an impact on quality of life that equal to or greater than that of the seizures themselves (Jacobs et al., 2009; Brooks-Kayal, 2011). Accordingly, the National Institute of Neurological Disorders and Stroke (NINDS) has identified the study of comorbidities as a major goal of current epilepsy research efforts (http://www.ninds.nih.gov/research/epilepsyweb/2007_benchmarks.htm.). Cortical dysplasia (CD) describes a range of malformations of cortical development and is a common cause of epilepsy in both children and adults. Developmental delay and cognitive impairment is a common comorbidity in CD, and there is some evidence that the severity of the impairment correlates with the extent of the cortical malformation (Barkovich & Kjos, 1992; Leventer et al., 1999). This study was undertaken as a first step to better understand how CD relates to impaired behavioral function in an animal model. We used in utero irradiation on embryonic day 17 (E17) to produce diffuse CD and examined the affected offspring using a battery of behavioral tests that monitor different aspects of brain function.

The effects of in utero irradiation on postnatal brain structure and behavior are dependent on both the timing and the dose of irradiation (Hicks & D'Amato, 1963; Brizzee, 1967; Norton & Kimler, 1987; Schull et al., 1990; Hossain et al., 2005; Kisková & Šmajda, 2006). Maximal effects on cortical development occur with irradiation between E15 and E18 (Cowan & Geller, 1960). Fetal irradiation can produce a wide range of structural defects including microcephaly, diffuse CD, neuronal heterotopia, ectopic pyramidal cells in the hippocampus, and agenesis of the corpus callosum (Riggs et al., 1956; Cowan & Geller, 1960; Roper et al., 1995). Irradiation can also cause reduced numbers of neurons in the cortex, hippocampus, and cerebellum (Brizzee et al., 1982; Norton & Kimler, 1987; Roper et al., 1995; Hossain et al., 2005; Schmitz et al., 2005; Zhou & Roper, 2010a), reduced numbers of dendrites and disorganized dendritic arborization (Brizzee et al., 1982), and defective neuronal connections (Rakic, 1988; Naylor et al., 2008).

CD is a common cause of medically intractable epilepsy (Taylor et al., 1971; Palmini et al., 1991), accounting for >50% of intractable epilepsy in children and up to 20% in adults (Kuzniecky & Barkovich, 1996). We have studied an animal model of CD by exposing fetal rats to 2.0–2.5 Gy γ-radiations on E17 (Roper et al., 1995, 1997), when cerebral cortex development is maximally sensitive to irradiation (Hicks & D'Amato, 1980). Postnatally, irradiated rats show spontaneous seizures in vivo (Kondo et al., 2001; Kellinghaus et al., 2004) and enhanced epileptiform activity in vitro (Roper et al., 1997). Previous electrophysiologic studies have demonstrated that an imbalance of synaptic input favors inhibition in interneurons in dysplastic cortex (Xiang et al., 2006; Zhou et al., 2009; Zhou & Roper, 2010b), but favors excitation in pyramidal neurons in dysplastic cortex (Zhu & Roper, 2000) and heterotopic gray matter (Chen & Roper, 2003). These findings complemented immunohistochemical studies that showed a selective reduction in the density of inhibitory interneurons (Roper et al., 1999; Zhou & Roper, 2010a,b) and γ-aminobutyric acid (GABA)ergic presynaptic terminals in this model (Zhou & Roper, 2010a). Impaired cortical inhibition and the subsequent increased excitation in these animals may be an important mechanism of epileptogenesis. But these widespread alterations in cortical and hippocampal circuitry may also have profound effects on other aspects of behavior and cognition.

Although previous studies have investigated behaviors in irradiated rats that were exposed to 0.3–1.5 Gy between E11.5 and E17.5 (Werboff et al., 1962; Norton, 1986; Norton & Kimler, 1987; Schull et al., 1990; Baskar & Devi, 1996, 2000; Devi et al., 1999; Kisková & Šmajda, 2006), the number of publications on behavioral effects of prenatal irradiation are still limited (Kisková & Šmajda, 2006). Not all time points and doses of irradiation have been examined, and the ages at testing are not always consistent. More importantly, although the behavioral effects of irradiation with relatively low doses have been investigated (Norton & Kimler, 1987), the behavioral effects of irradiation at higher doses, which has been demonstrated to produce CD with dyslamination (Roper et al., 1995, 1997), have not been reported. The present study was designed to investigate the behavioral effects of irradiation with 2.25 Gy on E17 on motor function, learning, and memory using a battery of behavioral tests. We found that rats with CD show impairment across multiple domains including motor coordination and both limbic-based and cortically based learning and memory.

Methods and Materials

Animals and irradiation

Pregnant Sprague-Dawley rats obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN, U.S.A.) were either sham-irradiated or whole-body irradiated with 2.25 Gy of external x-rays from a linear accelerator source on E17 (day of insemination, E0). Offspring from control pregnant rats were weaned on postnatal day 21 (P21), and offspring from irradiated pregnant rats were weaned at P28 due to development and growth delay. One-month-old male offspring from control (n = 8) and irradiated mothers (n = 14) were subjected to a series of behavioral tests. All rats were maintained on 12 h light/dark cycles and were given ad libitum access to food and water. All procedures used this study followed guidelines approved by the Institutional Animal Care and Use Committee at the University of Florida.

Activity and strength

Open field task

The open field task was used to assess general activity levels and overall locomotor activity (Botton et al., 2010). Each rat was placed in the center of the arena and was allowed to move freely for 5 min. The rat's behavior in the arena was monitored by an overhead video camera and was recorded using an automated tracking system (Ethovision, Noldus, The Netherlands) for offline analysis. Locomotor activity was tracked by the trace of the center-point of the body. General activity (including locomotor and nonlocomotor activity) was tracked by the trace of the nose-point of the body. Distance and time were measured to assess general and locomotor activities (Botton et al., 2010).

Grip strength task

Forelimb and hind limb grip strength, indicators of motor/neuromuscular function (Anderson et al., 2005), were determined using an automated grip strength meter with a horizontal forelimb or an angled hind limb mesh pull bar assembly (Columbus Instruments, Columbus, OH, U.S.A.). The mean force in grams was determined with a computerized electronic pull strain gauge that was fitted directly to the grasping mesh. Since body weight influences grip force, grip strength was expressed as the ratio of mean force (g) to body weight (kg). After allowing the animal to establish a good grip on the pulls bar, the experimenter gently pulled the rat away until its grasp was broken (Carter et al., 2009). The mean force was obtained. The test was repeated three consecutive times within the same session and the averaged value was recorded as the forelimb or hind limb grip strength.

Grid walk task

The grid walk test (foot fault test) was used to analyze sensorimotor coordination and the ability of the animal to perform coordinated forelimb and/or hind limb placement (Pazaiti et al., 2009). The grid walk apparatus consisted of an elevated 30-cm wire grid bridge that measured 120 × 30 cm and contained a dark escape box at one end. Three experimenters observed the rat's progress across the bridge and counted the number of the foot faults; the numbers were then averaged. Total time to cross the bridge was also recorded. Foot faults per min were used to estimate performance (Pazaiti et al., 2009).

Cognitive function

Object recognition task

The apparatus for the object recognition task consisted of objects and a square arena; the floor space of square arena was 60 × 60 cm and its walls were 60 cm in height (Fig. 1A). The walls were transparent and black curtains were used to avoid anxiety-provoking situations and cues in the room environment. The rats' behavior in the arena was monitored by camera and video recorded using the same setup as in the open field task. The task consisted of a sample phase and a test phase delivered 24 h later (Blalock et al., 2003). In the sample phase, two identical objects were placed in the forward left corner (object 1) and in the back right corner (object 2). The rats were allowed to explore the arena and objects for 5 min and then removed from the arena for a 24 h retention delay. For the retention test, one of the objects was replaced by a novel object. The rats were allowed to explore the arena and objects for 3 min and the time that the rat spent exploring each object was measured. Exploration of an object was defined as directing the nose <2 cm to the object. The exploration ratio was defined as ratio of time spent in exploring the novel object to total time spent in exploring both the novel and the familiar objects during the testing phase (TNovel/[TFamiliar + TNovel]) (Mumby et al., 2002; Bevins & Besheer, 2006).

Figure 1.

Figure 1

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Time engaged in object exploration and the exploration ratio for the objects during the object recognition task. (A) Schematics illustrate spent in exploring identical object 1 and 2 (O1 and O2) for 5 min during the sample phase. (C) Time spent in exploring the familiar object 1 and the novel object 1 (O1 and N1) for 3 min during test phases. Control and irradiated rats spent similar time by exploring the two identical objects 1 and 2 during the sample phase (B), whereas they spent much more time exploring the novel object 1 than the familiar object 1 during the test phase (C). (D) The exploration ratio for the objects decreased in irradiated rats. The ratio was obtained from TNovel 1/(TObject 1 + TNovel 1). The dashed line represents the chance level of performance (i.e., a ratio of 0.5). The rats in both control and irradiated rats spend more time in exploring the novel objects than the familiar objects (both groups the mean ratio >0.5). However, the difference between exploring familiar and novel object in control rats was greater than in irradiated rats. **p < 0.01 and*p < 0.05 versus control; ##p < 0.01 and #p < 0.05 versus O1 (object 1); $p < 0.01 versus control; &p < 0.01 in control and irradiation group, one sample t-test was performed.

Inhibitory avoidance task

The inhibitory avoidance apparatus (Coulbourn Instruments, Allentown, PA, U.S.A.) consisted of light and dark compartments separated by a sliding door; the floor consisted of a metal grid through which a mild shock was delivered (Fig. 2A). During acquisition training, the rat was placed in the lighted compartment for 90 s; the sliding door was then automatically opened. The rats quickly entered the dark compartment to avoid the bright light. The acquisition latency was defined as the time it took for the rat to enter the dark compartment. Once the rat entered the dark side, the door closed immediately; following a 10 s delay, a mild foot shock (0.21 mA for 3 s) was applied. The rat was then returned to the home cage. Twenty-four hours later, the rat was placed in the lighted chamber for 90 s and the door was opened. The retention latency (the time taken to enter the dark compartment) was recorded. If a rat did not enter the dark compartment within 900 s, the retention test was terminated and a retention latency of 900 s was assigned.

Figure 2.

Figure 2

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The latency to crossover to the shock-associated chamber was decreased in irradiated rats. (A) Schematics illustrate the general procedure for inhibitory avoidance task. (B) Latency in day 1 acquisition and day 2 retention testing. $p < 0.01 versus acquisition training of control rats; #p < 0.05 versus retention training of control rats.

Morris water maze task

The water maze has been utilized widely to assess hippocampal-dependent spatial memory and learning (Norris & Foster, 1999; Foster et al., 2003; Carter et al., 2009). The tank was 110 cm in diameter and was located in a well-lit room. A circular escape platform (13 cm in diameter) was positioned either just above (cue task) or below (spatial task) the surface of the water (27 ± 0.5°C). The animal's activity was monitored by an overhead video camera and was recorded using an automated tracking system (Ethovision).

Procedures for cue and spatial discrimination tasks have been published previously (Foster et al., 2003; Carter et al., 2009). For cue discrimination, a white flag was attached to the platform that extended above the water surface by 12 cm and the platform was extended 1 cm above the water level (Fig. 3A1). Curtains enclosed the pool and were used to limit spatial cues in the room environment. Prior to training, rats were habituated to the pool through a 30-s free swim and four trials to climb onto a platform from four different directions. Animals were then given five blocks of three consecutive trials of cue training for a total of 15 trials. Intertrial intervals were 20 s and interblock intervals were 15 min. The platform and start locations were randomized across each trial. The rats were allotted 60 s to escape during each trial; if they did not escape within the allotted time, they were gently guided to the platform where they remained for the intertrial interval.

Figure 3.

Figure 3

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Behavior is altered in irradiated rats in the water maze. Mean latency (A1), mean path length (A2),and velocity (A3) to escape during cue discrimination training. Mean latency (B1), mean path length (B2), and velocity (B3) to escape during spatial discrimination training. Each block consisted of three training trials. Control rats showed a significant decrease in the escape latency and path length during the five-block cue and spatial discrimination training; however, irradiated rats did not. Controls showed significantly shorter escape latency and path length than irradiated rats. The swim velocity was similar during training in the two groups.

Spatial discrimination training was performed 72 h after the cue discrimination task. For spatial discrimination, the curtains were removed to permit access to spatial cues in the room. The escape platform without an attached flag was submerged 1.0 cm beneath the surface of water (Fig. 3B1). The platform remained in the same location relative to the distal cues in the room; however, start locations were randomized across each trial. The rats were given six blocks of three trials. A probe trial was inserted between the fifth and sixth blocks of training in order to assess acquisition. Following this probe trial, a “refresher” block of three trials was provided. The refresher block was not included in statistical analyses that assessed learning. On the day following training, a second probe trial was employed to measure 24-h retention of the spatial discrimination. During the probe trials (Fig. 4A, inset), the platform was removed and the rat was allowed to swim for 60 s. For analysis of probe trial data, the number of times the rat crossed over the previous location of the platform was counted. Time spent in the four quadrants was also assessed. Latency to escape (seconds to reach the platform), swim velocity (cm/s), and distance (centimeters traveled in maze) were measured for both cue and spatial discrimination tasks.

Figure 4.

Figure 4

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Dwell time for goal quadrants and platform crossings are decreased in irradiated rats. Dwell time for goal quadrant and other quadrants of the water maze during acquisition (A) and retention probe trials (B) of spatial discrimination. Inset in A illustrates the location of goal quadrant (quadrant 3) and other quadrants. Control rats spent significantly more time in the goal quadrant than the other three quadrants in acquisition and retention probe trials but irradiated rats did not. The dashed line represents the chance level of performance. Platform crossings during acquisition (C) and retention (D) probe trials showed similar results. $p < 0.01 versus Q1, Q2, and Q4 in control rats; #p < 0.01, one sample t-test; *p < 0.01 versus control.

Histology

For cresyl violet staining, rats were deeply anesthetized (sodium pentobarbital, 60 mg/kg, i.p.) and perfused intracardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). After cryoprotection with 30% sucrose solution, frozen coronal sections (30 μm) containing cortex and hippocampus were obtained using a cryostat. Cresyl violet staining was performed using standard techniques.

Statistical analyses

All values are expressed as mean ± standard error of the mean (SEM). Specific methods of statistical analyses are described in the text. For all tests, p < 0.05 was considered statistically significant.

Results

Irradiation effects on development and growth, general activity, and motor function

Brain and body weight decrease and the brain-to-body weight ratio decrease in irradiated rats

The mean brain weight and body weight were significantly decreased in irradiated rats; the averaged ratio of brain-to-body weight also decreased significantly (all p < 0.01, Table S1).

General activity decreases in irradiated rats

The distance for general activity was significantly shorter in irradiated rats than in control rats (p < 0.01, Fig. S1A). The velocity of general activity was significantly decreased (p < 0.01, Fig. S1C) in irradiated rats. Distance and velocity for locomotor activity were not different between the two groups (both p > 0.05, Fig. S1B,D). Both control and irradiated rats kept moving for approximately 35% of the time using this measure.

Grip strength is not changed in irradiated rats

Mean grip strength was adjusted according to body weight (Carter et al., 2009). The forelimb and hind limb grip strength were 1422.7 ± 150.9 and 639.1 ± 21.4 g/kg [mean force (g)/body weight (kg)], respectively, in control, and 1264.9 ± 74.0 and 685.8 ± 46.7 g/kg, respectively, in the irradiated rats. These data showed that the irradiation affected neither forelimb nor hind limb grip strength (all p > 0.05), suggesting normal muscle strength or neuromuscular function in irradiated rats.

Grid walk performance is impaired in irradiated rats

The irradiated rats exhibited a significantly increased number of foot faults in the grid walk compared with control rats (p < 0.01). The mean foot faults were 2.78 ± 0.78 and 9.12 ± 1.58 faults/min, respectively, in control and irradiated rats. Those results suggest poor sensorimotor coordination in irradiated rats.

Irradiation effects on cognitive functions

Object recognition memory is impaired in irradiated rats

A two-way analysis of variance (ANOVA) on the time spent exploring identical objects during the sample phase indicated no object preference, such that rats spent an equal amount of time exploring each object. However, there was a significant group effect (F1,40 = 27.55, p < 0.00001), with the irradiated rats spending less time exploring objects relative to the controls (Fig. 1B). A two-way ANOVA examining the time spent exploring a familiar and a novel object during the test phase revealed a significant effect of group (F1,40 = 24.15, p < 0.00005) and object (F1,40 = 26.50, p < 0.00001) and a group × object interaction (F1,40 = 5.50, p < 0.05). Scheffe post hoc test indicated that control rats spent significantly more time than the irradiated animals spent exploring the novel object (p < 0.00005, Fig. 1C). We used the exploration ratio to estimate differential exploration activity. Irradiated rats had a significantly lower exploration ratio (p < 0.01, one-way ANOVA, Fig. 1D) than controls. Finally, we performed one-sample t-tests to determine whether the exploration ratios were significantly different from chance (chance = 0.5). The ratio was significantly above chance in control and irradiated rats (all p < 0.01, Fig. 1D). These results indicated that although irradiated rats were able to discriminate familiar and novel objects, cortex-dependent object recognition memory was significantly impaired.

Retention of inhibitory avoidance is impaired in irradiated rats

Mann-Whitney U tests indicated that the latency to cross to the dark compartment during the acquisition phase was not different between groups (Fig. 2B), suggesting that irradiated rats were able to discriminate between the two compartments and the groups did not exhibit differences in motivation to escape from the light. In contrast, Mann-Whitney U tests on the latency for retention testing revealed a significant group difference (p < 0.05, Fig. 2B) due to a decreased latency for irradiated rats relative to control rats. Paired t-test on the latency between the acquisition and testing phases revealed that control rats demonstrated an increased latency to cross to the dark compartment during the retention phase (p < 0.005). In contrast, no difference was observed for the irradiated rats (p = 0.26, Fig. 2B). These results indicated a poor retention ability or amnesia to avoid an aversive stimulus in irradiated rats.

Spatial memory and learning are impaired in irradiated rats

Cue discrimination task

The cue discrimination version of the water escape task can be employed to examine sensorimotor function (Fig. 3A1). For this task, we measured the latency and path length to escape from the water over blocks of training trials. A two-way ANOVA on escape latency revealed a significant main effect of group (F1,320 = 39.0, p < 0.00001) and block (F4,320 = 8.39, p < 0.00001) in the absence of an interaction. Examination of performance in each group indicated that control rats showed a significant decrease in latency across the five blocks of training (F4,115 = 9.50, p < 0.00001); however, the irradiated rats did not (F4,205 = 2.08, p = 0.086). The mean swim velocity for all five blocks was approximately 11 cm/s in both control and irradiated rats (Fig. 3A3), which did not contribute to the significant difference of the latency between the two groups and the blocks. Examination of the escape path length indicated a significant main group (F1,320 = 33.42, p < 0.00001) and block effect (F4,320 = 10.58, p < 0.00001) and no significant group-by-block interaction (F4,320 = 0.85, p > 0.05) (Fig. 3A2). Moreover, the escape latency was not significantly different on the first block of training, suggesting normal vision and swimming ability in irradiated rats.

Spatial discrimination task

Similar to cue discrimination, examination of escape latency for the spatial discrimination task indicated a significant main effect of group (F1,320 = 31.5, p < 0.00001) and block (F4,320 = 7.41, p < 0.00001), in the absence of an interaction (Fig. 3B1). Further repeated-measures one-way ANOVA showed a significant decrease in latency across the five blocks of training in controls (F4,115 = 4.57, p = 0.00184), whereas the irradiated rats did not (F4,205 = 1.89, p = 0.11363). ANOVA for the escape path length confirmed a significant main effect of group (F1,320 = 40.09, p < 0.00001) and training across blocks (F4,320 = 5.56, p < 0.0005) and no interaction effect (F4,320 = 1.14, p > 0.05) (Fig. 3B2). Again, repeated-measures ANOVAs within each group indicated a significant decrease in path length across the five blocks of training in controls (F4,115 = 8.63, p < 0.00001), whereas training effects were not observed for irradiated rats (F4,205 = 1.07, p = 0.37003). The mean swim velocity during spatial discrimination training was approximately 11 cm/s, similar to that during cue discrimination training in both control and irradiated rats (Fig. 3B3). The escape latency did not differ between the two groups on the first block of training. These results indicate that the irradiated rats exhibit impaired learning on the spatial discrimination task.

Probe trials

An ANOVA for the time spent searching the goal quadrant on the acquisition probe trial revealed that control rats spent significantly more time in the goal quadrant (quadrant 3) than did the irradiated rats (F1,20 = 11.59, p < 0.005, Fig. 4A). One sample t-tests were employed to determine whether the rats spent significantly more time in the goal quadrant than expected by chance (i.e., 15 s). The analysis revealed that only control rats spent significantly longer than 15 s in the goal quadrant (p < 0.01, Fig. 4A). An ANOVA on platform crossing confirmed that the irradiated rats exhibited impaired localization of the platform location relative to controls (p < 0.01, Fig. 4C), such that the number of platform crossings was reduced.

Similar to the acquisition probe trial task, during the retention probe trial task control rats spent significantly more time in the goal quadrant (quadrant 3) than did irradiated rats (F1,20 = 12.91, p < 0.005, Fig. 4B). Furthermore, only control rats spent significantly longer than 15 s in goal quadrant (p < 0.01). An ANOVA confirmed an impaired localization of the platform in irradiated rats as evidenced by the decreased number of platform crossings in the retention probe trial task (p < 0.01, Fig. 4D).

Histologic alterations in irradiated rats

A well-organized laminar distribution of neurons in the neocortex and hippocampus was observed in control rats (Fig. 5A). Irradiated rats showed thinning of the neocortex with loss of normal laminar organization. The hippocampi consistently showed focal areas of dispersion of the CA1 pyramidal cell layer (Fig. 5B). These observations are consistent with those of previous studies that describe in detail the histologic alterations in irradiated rats (Roper et al., 1995).

Figure 5.

Figure 5

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Cresyl violet-stained section of control and irradiated rats. (A) Low- (left) and high-power (right) images show well organized, six-layered neocortex and hippocampus. (B) The low-power image (left) shows thinning of the neocortex and focal areas of dispersion of the CA1 pyramidal layer. The high-power image (right) shows disorganized arrangement of neurons in the neocortex and better detail of hippocampal heterotopia (arrows). High-power images are indicated by the boxes in the low-power images. Scale bars for low-power images equal 300 μm and for high power images equal 100 μm.

Discussion

Developmental delay and impaired cognition are common comorbidities in people with CD-associated epilepsy. This study was undertaken to better understand the relationship between the structural abnormalities that have been characterized in irradiated rats and the behavior of the affected animals. Due to the diffuse nature of the structural abnormalities (including microcephaly, diffuse CD, and heterotopic neurons in the hippocampus and cortex), it is not surprising that a number of tests that rely on cortical and hippocampal function were abnormal in irradiated rats. However, a number of important functions (e.g., locomotor activity and vision) were preserved. Specific findings from the different tasks are described below.

Alteration of brain weight

The brain-to-body weight ratio in irradiated rats has been shown to be unchanged in one previous study (Devi et al., 1999). However, we found a significantly decreased brain-to-body weight ratio, indicating that low brain weight may be associated with defects in both general growth and brain development. Microcephaly has been described in animals irradiated on E17 (Cowan & Geller, 1960; Roper et al., 1995). Body weight has been shown not to influence escape from water during the water maze task (Vorhees & Williams, 2006). The significantly low brain weight, as a result of radiation-induced stunting of brain development, may produce behavioral deficits in irradiated rats.

No change in locomotor activity

Impaired locomotor activity could influence the ability to move in water or on the arena floor and consequently influence the performance in tasks such as the water maze. Previous studies have shown that spontaneous locomotion in irradiated rats was significantly decreased when doses of 0.3 Gy or higher were administered during E10.5–E14.5 (Werboff et al., 1962; Baskar & Devi, 2000; Hossian & Devi, 2000); however, it did not change (Baskar & Devi, 2000), or increased (Werboff et al., 1962), when rats were irradiated from E15 to E20. We found that the locomotor activity of rats exposed to irradiation with 2.25 Gy on E17 was not altered. However, general activity (locomotor and nonlocomotor) was significantly decreased in irradiated rats, indicating that nonlocomotor activity was primarily affected.

No change in grip strength

Alterations of motor function could influence performance in behavioral tests, such as grid walk and water maze tests. We found no reduction of forelimb or hind limb grip strength in rats that were irradiated with 2.25 Gy on E17. These results suggest that muscular strength did not influence performance on the behavioral tasks used in this study.

Poor performance in grid walk

The foot fault test was used to assess motor coordination and to quantify the ability of the animal to perform coordinated forelimb and hind limb placement in irradiated rats. Irradiated rats showed a significant impairment in sensorimotor coordination as evidenced by more misplaced steps. Grid walk performance requires sensorimotor coordination and is maximally affected by irradiation at E15–E17 when the cerebral cortex is being assembled (Kimler & Norton, 1988). The poor grid walk performance of irradiated rats in the current study suggests that the diffuse CD that has been demonstrated in these animals (Roper et al., 1995) is correlated with a quantifiable behavioral deficit.

Impaired object recognition performance

Object recognition memory corresponds to the ability to discriminate between novel and familiar objects and consists of at least two components: recollection and familiarity (Squire et al., 2007). Recollection is a conscious and effortful process in which the specific contextual details associated with an item are retrieved. Familiarity is a relatively fast and automatic process in which one senses having encountered an item without retrieving any specific details (Wais et al., 2010). It is still not clear which brain regions are directly involved in recollection and familiarity (Muñoz et al., 2010); however, it has been proposed that both recollection and familiarity of recognition memory rely mostly on cortical areas (including prefrontal cortex and lateral parietal cortex and perirhinal cortex) (Yonelinas et al., 2005; Haskins et al., 2008). The role of the hippocampus in recognition is unclear. Some studies have shown that the hippocampus is involved in recollection (Yonelinas et al., 2005; Wais et al., 2010), whereas others have suggested that the hippocampus is not essential for object recognition in this paradigm (Mumby et al., 2002). The effect of in utero irradiation on object recognition had not been fully investigated. One previous study showed that short-term exploration of a novel environment, using a continuous corridor apparatus or home cage emergence, is suppressed by irradiation with 0.5 Gy and above on E15 or E17 (Schull et al., 1990). The current study revealed an impaired object recognition performance in irradiated rats, consistent with our previous observation showing an alteration of cortical structure (Roper et al., 1995; Zhou & Roper, 2010a).

Impairment in inhibitory avoidance

Inhibitory (passive) avoidance task is a behavioral paradigm whereby an animal learns to inhibit a response in order to avoid an aversive stimulus such as electric shock. Inhibitory avoidance procedures produce rapid and enduring learning, and noninjurious foot-shock can produce a retained fear memory for days or even months (Maren, 2008). The amygdala has been shown to be critical for learning about discrete, unconditioned stimuli such as foot-shock (Phillips & LeDoux, 1992).Good performance in inhibitory avoidance task is amygdala dependent (Giese et al., 2001). For an animal to associate an aversive stimulus the hippocampus must be intact, and thus the hippocampus also influences inhibitory avoidance task performance (Rogan & LeDoux, 1996). Previous studies have shown that irradiation can cause an abnormal hippocampus (Roper et al., 1995; Hossain et al., 2005; Schmitz et al., 2005), but effects on the amygdala are unclear. The hippocampus and amygdala dynamically complement each other and interact with respect to memory and learning (Richter-Levin & Akirav, 2000). Therefore, the impaired inhibitory avoidance memory and learning in irradiated rats can be the consequence of an abnormal hippocampus or amygdala or hippocampus-amygdala interactions. A previous study in irradiated rats showed a significant impairment in contextual fear conditioning (Tamaki et al., 1989), a hippocampus-dependent learning task (Liu et al., 2004).

Impaired spatial memory and learning

Cued and spatial tasks can be used to test for learning ability in the Morris water maze. Both tasks require the same basic motor skills and strategies, and the same motivation (Vorhees & Williams, 2006). We feel that the failure of irradiated rats to learn the cue tasks indicates an impairment of overall learning rather than impairment of basic abilities such as eyesight, swimming, or motivation to escape to the platform. First, irradiated rats appeared to have intact vision. The irradiated rats found the visible escape platform, even though they had a longer latency than control rats did during the cue task. Irradiated rats also spent more time exploring novel objects than the familiar one during the object recognition task, another measure of visual ability (Save et al., 1992; Thinus-Blanc & Foreman, 1993). Second, the irradiated rats swam with the same speed as the control rats. Third, although motivation is difficult to measure directly, the irradiated rats swam as vigorously as the controls and tried to escape the water by jumping onto the wall of the tank. For these reasons, we feel that poor performance on the water maze tasks represents a true impairment of learning and memory in irradiated rats.

Irradiation effects on learning and memory in animals are dependent on both the timing of irradiation (in relation to brain development) and the dose of irradiation. Irradiation with doses >0.3 Gy produce detectable behavioral changes and increased doses may increase irradiation effects. Irradiation during E10-E18 has been reported to impair learning and memory (Devi & Baskar, 1996; Devi et al., 1999; Baskar & Devi, 2000), but some reports have provided contradictory findings. Irradiation at E13 and E15 has been reported to produce no significant effect on learning and memory during spatial tasks (Sienkiewicz et al., 1999; Kisková & Šmajda, 2006). The age at testing may be also an important factor that influences the behavioral effects of irradiation on postnatal behavior. Exposure to 0.50 Gy of γ-radiation at E11.5 was reported to induce behavior changes in 12-month-old mice but not in 18-month-old mice. Our study showed that irradiation with 2.25 Gy at E17 significantly impaired spatial memory and learning when testing 1-month-old rats. These results suggest that the hippocampus was affected by irradiation, and this has been documented in several histologic studies (Roper et al., 1995; Hossain et al., 2005; Schmitz et al., 2005).

In conclusion, this study has documented major impairments in learning and memory that depend on the cortex and hippocampus in rats with diffuse malformations of cortical development. One confounding factor in this study could be the effect of seizures on cognitive performance. Rats irradiated on E17 have been reported to have rare seizures (Kondo et al., 2001; Kellinghaus et al., 2004). However, in one study (Kondo et al., 2001), chronic seizure frequency was low (0.01 seizures/24 h). In the other study (Kellinghaus et al., 2004), rare chronic seizures (0.57 seizures/24 h) were seen in rats that received moderate doses of radiation (145 cGy) but not at higher does (175 cGy). These data would suggest that it is unlikely that the animals in the current study were experiencing spontaneous seizures during the testing period. Although no video or electroencephalography (EEG) recordings were taken for this study, the animals were observed closely during the days of testing and no recognizable seizures were detected. This does not completely rule out the possibility of “subclinical” seizure activity during testing or seizures that occurred overnight between acquisition and retention testing. But the fact that seizures are rare in this model and that the cognitive defects were seen consistently throughout the irradiated animals over several days of testing makes this potential confounder unlikely.

This study has shown that animals with diffuse abnormalities of cortical and hippocampal development have severe impairment of learning and memory subserved by those brain structures. This sets the ground work for future studies to examine the mechanisms of these impairments and to evaluate the efficacy of potential future therapeutic interventions.

Supplementary Material

FigS1

Figure S1. The general activity is reduced in irradiated rats.

TableS1

Table S1. Brain and body weight and the ratio of brain-to-body weight.

Acknowledgments

This work was supported by the Citizens United for Epilepsy Research (CURE), the McKnight Brain Research Foundation, and the Densch Foundation.

Footnotes

Disclosure: The authors have no conflicts of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Supporting Information: Additional Supporting Information may be found in the online version of this article:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FigS1

Figure S1. The general activity is reduced in irradiated rats.

NIHMS657902-supplement-FigS1.tif (56.7KB, tif)
TableS1

Table S1. Brain and body weight and the ratio of brain-to-body weight.

NIHMS657902-supplement-TableS1.pdf (95KB, pdf)

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