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pilepsy Research (2014) 108, 1032—1040

j ourna l h om epa ge: www.elsev ier .com/ locate /ep i lepsyres

pecific alterations in the performance ofearning and memory tasks in models ofhemoconvulsant-induced statuspilepticus

ennifer N. Pearsona, Kalynn M. Schulzb,d, Manisha Patela,c,∗

Neuroscience Program, University of Colorado Anschutz Medical Campus, United StatesDepartment of Psychiatry, University of Colorado Anschutz Medical Campus, United StatesDepartment of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, United StatesMedical Research Service, Veterans Affairs Medical Center, Denver, CO, United States

eceived 18 November 2013; received in revised form 2 April 2014; accepted 19 April 2014vailable online 29 April 2014

KEYWORDSChemoconvulsant;Kainic acid;Pilocarpine;Epileptogenesis;Novel objectrecognition;Learning and memory

Summary Cognitive impairment is a common comorbidity in patients with Temporal LobeEpilepsy (TLE). These impairments, particularly deficits in learning and memory, can be reca-pitulated in chemoconvulsant models of TLE. Here, we used two relatively low-stress behavioralparadigms, the novel object recognition task (NOR) and a spatial variation, the novel place-ment recognition task (NPR) to reveal deficits in short and long term memory, in both kainic acid(KA) and pilocarpine (Pilo) treated animals. We found that both KA- and Pilo-induced significantdeficits in long term recognition memory but not short term recognition memory. Additionally,KA impaired spatial memory as detected by both NPR and Morris water maze. These deficits were

present 1 week after SE. The characterization of memory performance of two chemoconvulsant-models, one of which is considered a surrogate organophosphate, provides an avenue for whichtargeted cognitive therapeutics can be tested.© 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author at: 12850 E. Montview Boulevard V20-238, Aurora, CO 80045, United States. Tel.: +1 303 724 3604.

E-mail address: Manisha.Patel@ucdenver.edu (M. Patel).

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ttp://dx.doi.org/10.1016/j.eplepsyres.2014.04.003920-1211/© 2014 Elsevier B.V. All rights reserved.

ntroduction

ognitive impairment is a common co-morbidity of the

pilepsies and is especially pronounced in patients withedically refractory seizures and patients with Temporal

obe Epilepsy (TLE). These impairments, including deficitsn declarative and spatial memory (Abrahams et al., 1999;

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Chemoconvulsants and learning and memory

Guerreiro et al., 2001; Ploner et al., 2000; Viskontas et al.,2000; Elger et al., 2004), contribute significantly to the dis-ability experienced by people with epilepsy. While manyclinicians associate cognitive dysfunction specifically withTLE, there is increasing awareness that these deficits arealso common in people with other epilepsy syndromes,including extratemporal (Adams et al., 2008) and geneticgeneralized epilepsies (Adams et al., 2008; Akanuma et al.,2008; Christensen et al., 2007; Filho et al., 2011; Hermannet al., 2008). Whether cognitive deficits are merely theside effect of medication, arise from the seizures them-selves, or share a common underlying etiology is the topicof much speculation. Additionally, the threat of progressivecognitive impairment resulting from frequent uncontrolledseizures (Pitkanen and Sutula, 2002) underscores the needfor further research on mechanisms of cognitive dysfunctionassociated with epilepsy. Such research is complicated in thehuman due to a multitude of factors including genetic back-ground, medication status and history, and other comorbidconditions such as anxiety and depression, any of which cannegatively impact cognitive function. Animal models mayafford the greatest opportunity to dissect out the roles eachof these play in contributing to cognitive impairment asso-ciated with epilepsy.

A wide variety of animal models have been developedfor the study of specific types of epilepsy (reviewed inSarkisian, 2001) and although no single animal model canprecisely mimic the human condition, several recapitulateimportant facets of the disease. Perhaps the most commonlystudied experimental models of TLE are the chemocon-vulsants, kainic acid (KA) and pilocarpine (Pilo). Systemicadministration of either KA (an analog of glutamate) or Pilo(a cholinergic agonist) results in a characteristic patternof intense limbic seizures typically culminating in statusepilepticus (SE) or a period of continuous seizure activity.Animals that experience SE-induced by either KA or Pilo arelikely to go on to develop spontaneous seizures and becomeepileptic (Hellier et al., 1998). Chemoconvulsant-inducedneuropathology is very similar to TLE-related neuropatho-logy and includes neuronal loss, gliosis, mossy fiber sproutingand synaptic reorganization (Ben-Ari, 1985).

Chemoconvulsant TLE models offer advantages for study-ing cognitive dysfunction associated with epilepsy. First, useof animal models allows for the identification of potentialmechanisms of seizure-induced cognitive damage. Secondly,the chemoconvulsant models allow preclinical testing oftherapeutic candidates for cognitive dysfunction occurringin acquired epilepsy (Brooks-Kayal et al., 2013). Addi-tionally, chemoconvulsants such as Pilo, mimic long-termneurochemical and behavioral changes occurring followingexposure to chemicals such as nerve agent and/or metabolicpoisoning (Jett, 2010). In fact, Pilo has been used as a surro-gate of nerve agent neurotoxicity and seizure activity plays amediating role in its long term toxicity. Therefore the use ofchemoconvulsant models to study seizure-induced cognitivedysfunction may yield therapeutic targets beyond TLE.

Behavioral testing in seizure-prone animals represents aseries of unique challenges to both selection of paradigms

and timing of task performance. Some of the most com-monly used paradigms to evaluate learning and memoryinclude the Morris Water Maze (MWM), the Radial Arm Maze(RAM), Contextual Fear Conditioning (CFC), and Delayed

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on-Matching to Sample (DNMS). These paradigms haveeen used to reveal learning and memory deficits in vari-us models of epilepsy including chemoconvulsant-inducedE models, kindling models and some genetic models. How-ver, the use of these tasks to screen for targeted therapiesgainst cognitive deficits associated with the epilepsies maye problematic. First, the use of aversive techniques (water,hocks, and food restriction) to spur performance in thesearadigms introduces a possible confound of stress. Fur-hermore, any drug that affects stress responses or anxietyevels may improve performance and be interpreted asmproving learning and memory, when it simply functionss an anxiolytic. Secondly, stress is among the most fre-uently self-reported precipitants of seizures in patientsith epilepsy (Frucht et al., 2000; Spector et al., 2000;akken et al., 2005; Haut et al., 2007), so it stands to reasonhat behavioral tasks that elicit stress may induce seizuresn animals with a lowered seizure threshold. It is thereforedeal to include in the cognitive testing battery, tasks thatre low-stress to get an accurate representation of learningnd memory performance.

The current study tested the effects of two chemocon-ulsants, KA and Pilo, on learning and memory performancesing two minimally stressful variants of the novel objectecognition paradigm, the novel object recognition taskNOR) and the novel placement recognition task (NPR). Welso measured locomotion and anxiety-related behavior inhe open field in order to assess these parameters duringhe latent period and also to determine if these factors couldccount for differences in learning and memory. Finally, wemployed the Morris Water Maze (MWM) to allow for com-arisons between memory paradigms.

aterials and methods

nimals

ale Sprague-Dawley rats (250—300 g) were purchased fromarlan Laboratories (Indianapolis, Indiana). Upon arrival,nimals were housed two per cage in static clear polycarbo-ate cages with wire bar lids and microisolator air filtrationovers. Animals had ad libitum access to both food and fil-ered water. Room conditions were maintained at 21 ◦C with

14:10 light/dark cycle. Animals were treated in accor-ance with NIH guidelines and all protocols were approvedy the IACUC of the University of Colorado Denver.

hemoconvulsant injections

ll animals were handled for approximately 2 min per daytarting a week before treatment both to accustom thenimals to the investigator and to potentially reduce anytress associated with handling on subsequent testing days.n the day of treatment, animals were randomly assignedo either the control or experimental (KA or Pilo) group.he experimental groups were administered injections ofhe chemoconvulsants kainic acid (KA, 11 mg/kg, subcuta-

eously; s.c. Sigma—Aldrich) or Pilocarpine hydrochloride340 mg/kg, s.c. Sigma—Aldrich) in buffered PBS. Animalsreated with Pilo were injected with scopolamine (1 mg/kg,P) 30 min prior to Pilo to limit peripheral cholinergic effects

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nd diazepam (10 mg/kg, IP) 90 min after Pilo to termi-ate SE (as is the standard of procedure for a cholinergicgonist to reduce mortality). Control animals for the Piloroup were injected subcutaneously with scopolamine, andqual volumes of saline in place of Pilo and diazepam. Con-rol animals for the KA group received a single injectionf saline in place of KA. After treatment, the experimen-al groups were observed for the characteristic progressionnto SE. Chemoconvulsant administration typically elicitseizures of increasing severity starting with staring and wetog shakes and then progressing into unilateral forelimblonus, bilateral forelimb clonus before reaching the mostevere seizures which result in rearing and loss of balanceTremblay et al., 1984; Sperk, 1994). Ultimately, administra-ion of chemoconvulsants results in a period of continuouseizure activity for at least 30 min known as SE. Only animalshat progressed into SE as defined by five seizures resulting inilateral forelimb clonus and loss of balance within an hourRacine, 1972) followed by a period of continuous seizurectivity were included in subsequent studies. The day fol-owing treatment, all animals received a 1 ml subcutaneousnjection of saline to help prevent any dehydration the ani-als treated with chemoconvulsants may have experienced.

reatment occurred at 77 days of age and is considereday 0 for the behavioral testing timeline. All animals werebserved, yet left undisturbed for a recovery period of 3ays.

ehavioral testing

pparatus and stimuliehavioral testing apparatus consisted of two identicalehavioral arenas constructed of mat black expanded PVC70 cm × 70 cm; wall height = 47.6 cm). A false floor madep of four removable PVC pieces were used to facilitateultiple spatial arrangements of the objects. The stimulus

bjects consisted of vinyl dog toys (Lil’ Buddies) that var-ed in color, shape, and texture but were of similar size andonstructed of the same material. Ten unique toy types weresed across testing days allowing for one toy type (or two inhe case of the novel object recognition task) to be used on aarticular test day and not be used again throughout testingor that particular animal. A pilot study of the objects, per-ormed with a separate cohort of animals, showed that nopecific object elicited more investigation than any other.o prevent objects from being dislodged by the animal, thebjects were zip-tied to inverted jars that screwed firmlynto place on individual segments of the false floor. Stimulusbjects were cleaned between all tasks with a disinfectant70% isopropyl alcohol) and an odor remover (Nature’s Mir-cle). The behavioral arena was cleaned between all tasksith a non-toxic deodorizing solution (Simple Green).

xperimental designn order to assess the learning and memory profile of ani-als treated with chemoconvulsants compared to untreated

ontrols, we utilized a battery of behavioral paradigms.

esting was performed on consecutive days across a timepan of about 1 week. After recovery from chemoconvulsant-nduced SE, animals were acclimated to testing parametershrough conditions approaching the experimental paradigms

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J.N. Pearson et al.

hat were to be used as highlighted in Fig. 1. Specifically,nimals were first exposed to the dedicated behavioral suitehere the entirety of the study was performed (room accli-ation). On the next day, animals were tested in an openeld for a period of 10 min in order to gage locomotor activ-

ty and indices of anxiety, this also served as the initialcclimation to the behavioral arenas. The following day,timulus objects were placed into the arenas and the ani-als were allowed to acclimate to the objects. Specificallyuring this ‘‘object investigation’’, animals investigated twodentical objects for a period of 10 min before being placedack into their home cages. Objects used during the toycclimation were not used again in any later testing. The 4ays following the toy acclimation, animals were subjectedo learning and memory testing. The behavioral testingonsisted of one task per day alternating between novellacement recognition (NPR) and novel object recognitionasks (NOR) and two delay lengths, 5 min and 1 h. A separateohort of animals was used for the NOR task at the 24 h delayNOR24) and Morris Water Maze tasks both to reduce theikelihood of carryover testing effects and to ensure that allasks were performed within two weeks of SE, when seizurerequency is low.

rocedurell behavior testing occurred at about the same time eachay (i.e. 9:00 am—3:00 pm) and after a period of at least 1 ho allow the animals to acclimate to the room. Test order wasandomized for each day of testing using a random numberenerator (random.org) and the arena tested in was coun-erbalanced across days for each animal. Whether the noveltimulus object for the NOR task was presented on the leftr right side of the behavioral arena was counterbalancedor each animal. All behavioral testing occurred during theight phase of the light/dark cycle by a single investigatorlind to group assignment.

ovel placement recognition taskriefly, this task consisted of two phases, a learning phasend a memory phase (Fig. 2). During the learning phase, ani-als were placed into the behavioral arena for a period of

min and allowed to explore two identical stimulus objectsefore being placed back into the home cage. After a delay,he animals were placed back into the arena where theyad the learning phase with the same stimulus objects,xcept during the memory phase, one of the objects was dis-laced to a novel spatial location. Cognitively intact animalshould notice this change and spend more time investigat-ng the displaced object as opposed to the object in theamiliar location. Previous studies have shown that this tasks hippocampal-associated (Mumby et al., 2002; Dix andggleton, 1999; Mumby, 2001). This task was performed oncet a delay length of 5 min (NPR5) and again at a delay lengthf 1 h (NPR60).

ovel object recognition taskimilar to the NPR task described above, the novel object

ecognition task (NOR) also consisted of a learning phasend a memory phase (Fig. 2). The task was performedxactly as described above with the exception of duringhe memory phase, rather than the target object being

Chemoconvulsants and learning and memory 1035

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Figure 1 Schematic of behavioral testing schedule. NPR — no— Morris water maze.

displaced to a novel spatial location, it was replaced with anentirely new stimulus object. Again, animals should noticethis change and spend more time investigating the novelobject as opposed to the familiar object. Previous stud-ies have shown that this task is not strictly hippocampal-dependent, but also depends on an intact perirhinal cortex(Mumby et al., 2002; Dix and Aggleton, 1999; Mumby, 2001;Kealy and Commins, 2011). As with the NPR trials, NOR wasperformed first at a delay length of 5 min (NOR5) followed bya delay length of 1 h (NOR60). A separate cohort of animalswas used for the 24 h delay and the MWM.

Morris water mazeThe water maze consisted of a tank approximately 6 feet indiameter and 2 feet deep. The water (room temperature)was colored with non-toxic black tempura paint to obscurea plexiglass platform that was located within the top leftquadrant of the tank. Animals were placed into the tank

from various positions that were randomized across trails foreach day. Surrounding the tank were various cues for naviga-tion including markings on each wall and three-dimensionalobjects hanging above the maze, all within eyesight of the

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Figure 2 Schematic of novel

placement recognition; NOR — novel object recognition; MWM

nimals. Each animal performed four trials per day for 5ays. A trial consisted of an animal being gently placednto the tank facing the inner wall. The animal was allowedo swim freely for 2 min or until it reached the platformhere it was allowed to rest for 15 s. If the animal hadot reached the platform in the allotted time its latencyas noted as 120 s and it was gently guided to the platformnd allowed to remain for 15 s. Latency to reach platformas measured and quantified using automated behavioral

oftware (Topscan, Clever Sys, Inc., Reston, VA) over tri-ls and days as an indicator of spatial learning. 24 h afterhe last trial on day 5, a probe test was performed tossess spatial memory. Time spent in the target quadrantthat formerly held the platform) was measured over a 30 seriod.

ehavioral analysesll tasks were video recorded and behaviors of interest

ere quantified using Topscan behavior recognition soft-are (Clever Sys. Inc, Reston, VA). Behavior parametersuantified for each task included: locomotor measures suchs distance traveled and velocity within the arena (open

object testing paradigm.

1036 J.N. Pearson et al.

Figure 3 Open field performance of KA and Pilo treated animals compared to controls. Significant differences were detectedbetween groups on measures of locomotion (A) as measured by total distance traveled, KA treated rats were hyperactive but (B)velocity was not different between groups. Indices of anxiety including the duration of time (c) and frequency of visits to thecenter of the arena (D) were equivalent between groups. A common indices of risk assessment (C), stretch-attend behaviors weredecreased in both KA and Pilo treated animals relative to controls, however, latency to the center of the arena was not differentb essed( t dif

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etween groups. Control, n = 10; KA, n = 9; Pilo, n = 7. Data exprp < 0.001) between groups, Pound sign (#) indicates a significan

eld, NOR, NPR), and investigatory measures such as timepent sniffing the stimulus objects and latency to sniff eitherbject (NOR, NPR). Investigation of the stimulus objectsas recorded as both frequency and duration and wasefined as any instance in which the animal’s nose wasriented within at least 4 mm of the object. From theseeasures, the proportion of total visits to the novel object

nd the proportion of total time spent investigating theovel object was calculated (novel/(novel + familiar inves-igation)). Previous studies have found that object noveltyuickly diminishes during the recognition phase (Dix andggleton, 1999; Mumby, 2001), so our analyses were focusedn the first 30 s of the recognition phase in learning and

emory testing. Data presented from the open field task

nd toy acclimation are the full 10 min in order to ensurehat there were no differences in locomotor activity thatould explain deficits in toy investigation during the learning

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as mean ± SEM. Asterisk (***) denotes a significant interactionference (p < 0.05) from control.

nd memory tasks. MWM data were analyzed for latency tolatform and swim speed/distance.

tatistical analysispen field data were analyzed using one-factor ANOVA tossess the effect of chemoconvulsants on locomotion andndices of anxiety. NOR and NPR tasks were analyzed using

two-factor repeated measures ANOVA using treatmentroup as the independent variable and task delay length ashe repeating variables. For the MWM, animals performedour trials per day for 5 days and performance of eachay was averaged and analyzed by means of a two-way

epeated measures ANOVA with treatment groups and serv-ng as the independent variable and day as the repeatingactor. Significant main effects and interactions were probedsing Bonferroni post-tests. Differences were considered

1037

Figure 4 Effects of chemoconvulsant induced SE on noveltypreference ratios on novel placement recognition trials. At the5 min delay, KA treatment significantly reduced the noveltypreference ratios compared to control, while treatment withPILO was not significantly different from control. At the 60 mindelay, a similar pattern was observed, treatment with KA signifi-cantly reduced novelty preference ratios compared to controls,while treatment with Pilo resulted in preference ratios not sig-nificantly different from control. Control, n = 10; KA, n = 9; Pilo,nn

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In order to evaluate predominantly perirhinal cortex-associated recognition memory, animals were subjected to anovel object recognition task at three delay lengths: 5 min,1 h and 24 h. Memory performance on these tasks, again

Figure 5 Effects of chemoconvulsant induced SE on noveltypreferences in the novel object recognition tasks at differentdelay lengths. Chemoconvulsant treatment did not significantlyaffect performance at the 5 min delay length for either KA orPilo groups. At the 60 min time point, treatment with KA sig-nificantly reduced novelty preferences, however, Pilo treatedanimals were not significantly different from controls. At the24 h delay, both KA and Pilo significantly reduced novelty pre-

Chemoconvulsants and learning and memory

significant when p ≤ 0.05. All statistical analyses were per-formed using GraphPad Prism 5. All data are expressed asmean ± SEM.

Results

Open field

To exclude the possibility that differences in mobility andanxiety could account for differences in exploratory behav-ior, these parameters were initially examined in an openfield task but monitored throughout testing. Interestingly,total distance traveled within the open field arena was sig-nificantly different between groups (Fig. 3a; F(1,27) = 8.853,p = 0.0012) such that KA-treated rats were slightly hyperac-tive relative to control (p < 0.05), however, velocity withinthe arena was similar across groups (Fig. 3b; F(1,27) = 1.879,p = 0.1737). This difference in total distance traveled did notpersist after the open field task, specifically the followingday, all animals were acclimated to objects and locomo-tion during this task, as measured by total distance traveledwas not significantly different between groups (data notshown). Additionally, indices of anxiety including frequencyand duration of time spent in the center of arena wasnot significantly different between groups (Fig. 3c and d;F(1,27) = 0.084, p = 0.9201 and F(1,27) = 1.779, p = 0.1894).Latency to the center of the arena was also not signifi-cantly different between groups (Fig. 3e; F(1,27) = 0.2048,p = 0.2048) Interestingly, both Pilo (p < 0.001) and KA groups(p < 0.05) had significantly reduced instances of stretch-attend behaviors (Fig. 3f; F(1,27) = 9.271, p = 0.001) whichare defined as instances when the animal stretches tosniff the center of the arena while the body is locatedin the perimeter, these postures are commonly associatedwith risk assessment (Pinel and Mana, 1989). Risk assess-ment behaviors such as stretch attends are investigatory innature and allow the animal to gain information about apotential threatening environment (Pinel and Mana, 1989).Taken together, these data indicate animals treated withchemoconvulsants do not have any differences in locomotionthat may account for differences observed in the learn-ing and memory tasks. Additionally, no differences wereobserved in measures of anxiety related behavior, i.e. fre-quency and duration in the center of the arena betweenchemoconvulsant-treated animals and controls, suggestingthat differences in the performance of the learning andmemory tasks are not due to differences in anxiety. Inter-esting, the data do suggest that risk assessment behaviormay be reduced in animals that experienced SE comparedto controls.

Novel placement recognition

To evaluate predominantly hippocampal dependent spatialmemory in chemoconvulsant animals, a novel object place-ment task was performed at two delay lengths: 5 min and1 h. Performance on this task varied depending on treatment

group (F(2, 23) = 10.87, p = 0.0005). Specifically, KA-treatedrats exhibited a lower novel object preference ratio at bothdelays (p < 0.05). A significant decrease in the proportionof time spent investigating the novel location is suggestive

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= 7. Data expressed as mean ± SEM. Asterisk (*) denotes a sig-ificant difference (p < 0.05) between groups.

f deficits in spatial memory. Finding deficits at both timeoints is indicative of significant and persistent deleteriousffects of KA-induced TLE on spatial memory. Interestingly,his deficit was not observed in the Pilo treated animals atither delay length (Fig. 4).

ovel object recognition

erences relative to control animals. Control, n = 10; KA, n = 9;ilo, n = 7. Data expressed as mean ± SEM. Asterisk (*) or (**)enotes a significant difference (p < 0.05 or p < 0.01, respec-ively) between groups.

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aried as a function of treatment group (Fig. 5; F(2,50)0.53, p = 0.0005). Post hoc analysis revealed significant dif-erences between control and KA groups at the 1 h delayp < 0.05) and at the 24 h delay, both KA and Pilo groups wereignificantly different than control (p < 0.01). Effect of delayength trended toward significance at p = 0.063. These datauggest that long-term recognition memory but not short-erm recognition memory is impaired in both KA and Piloodels.

orris water maze

o determine the extent to which spatial learning andemory was impaired in chemoconvulsant treated animals

ompared to controls, animals were tested in the Mor-is Water Maze (MWM). Latency to reach the submergedlatform varied as a function of treatment (Fig. 6A; F(1,8) = 69.07, p < 0.0001) and day (Fig. 6A; F(1, 88) = 25.6,

< 0.0001; interaction F(1,88) = 7.771, p < 0.0001). Post hocnalysis revealed a significant effect of KA treatment onatency to reach the platform on days 2, 3, 4 and 5 andor and a significant effect of Pilo treatment for animals onll days tested. This significantly greater amount of time toeach the platform than controls is indicative of deficits inpatial learning (D’Hooge and De Deyn, 2001). When testedn a probe trial for spatial memory 24 h after the last trial onay 5, significant differences were detected between groupsn the amount of time spent searching the target quad-ant (Fig. 6B; F(1,22) = 3.763, p = 0.0393), suggesting thatreatment with chemoconvulsants affects spatial memoryn this task. Interestingly, animals injected with KA or Pilout not experiencing SE showed deficits similar to saline-reated controls (data not shown), suggesting a role for SEs the initiating event in mediating cognitive deficits. Theseata suggest that chemoconvulsant-induced SE, regardlessf the type of chemoconvulsant, significantly and deleteri-usly affects spatial learning and memory.

iscussion

he primary goal of this study was to characterize the learn-ng and memory performance of chemoconvulsant-treated

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igure 6 Effects of chemoconvulsant induced SE on performanceffected latency to find a submerged platform using spatial cues. (Bent in Pilo and KA treated rats compared to control. Control, n = 10

***) denotes a significant difference (p < 0.001) between groups.

J.N. Pearson et al.

nimals. Two mechanistically distinct chemoconvulsants,A and Pilo, where chosen to initiate TLE and to revealreatment-induced memory deficits. Two variants of theovel object recognition test were employed, the NOR andPR tasks, both of which are minimally stressful. By employ-

ng multiple delay lengths in both NOR and NPR tasks, weere able to test both short-term and long term memory onach of these tasks. Spatial memory was also evaluated usinghe MWM, allowing for comparison of performance betweenwo distinct memory paradigms.

Both KA and Pilo animals exhibited significant deficitsn the long delays of the NOR task suggesting that expo-ure to such chemoconvulsants results in impaired long-termecognition memory. Interestingly, short-term recognitionemory (i.e. 5 min delay) was spared in both chemocon-

ulsant models on the novel object recognition task (NOR).dditionally, both Pilo and KA groups exhibited spatial learn-

ng deficits in the MWM, with Pilo animals being virtuallynable to learn the task. Despite the significant impair-ent in the MWM, Pilo-treated animals exhibited above

hance novelty preference ratios for the novel spatial loca-ion indicative of intact spatial memory on the NPR task. Its possible that cessation of Pilo-induced SE with diazepamay have protected against deficits on this task but to pre-

ent excessive mortality, the use of diazepam is typicallyhe standard. KA-treated animals on the other hand exhib-ted significantly reduced novelty preferences relative toontrols, indicative of spatial memory impairment. Theseata suggest that the NPR task may be effective in revealingpatial memory deficits in animals treated with KA but notnimals treated with Pilo. These results are not due to dif-erences in seizure susceptibility because all animals wentn to develop spontaneous seizures at equivalent observedrequency.

The finding of Pilo animals exhibiting intact spatial mem-ry on the NPR but not the MWM is of interest, particularlyiven the recently emerging interest in evaluating therapiesn models of organophosphate neurotoxicity. It is possiblehat the stress associated with the MWM, namely the shock

f swimming, compromises memory performance for thatask. Indeed, a common technique adopted by Pilo treatedats during MWM testing was thigmotaxis (wall hugging)hich can indicate increased anxiety and fear (Treit and

in the Morris Water Maze. (A) Both KA and Pilo significantly) A probe test for spatial memory revealed a significant impair-; KA, n = 9; Pilo, n = 7. Data expressed as mean ± SEM. Asterisk

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Chemoconvulsants and learning and memory

Fundytus, 1988). This behavior occurred even when the plat-form was visible, raising concerns about the ability of theanimals to participate in the task. Thigmotaxis and otherindices of anxiety were not observed in the open field taskperformed just days before the MWM indicating that it is spe-cific to the swimming task. It may therefore be beneficial totest spatial learning and memory in various tasks, includ-ing the NPR task to get an accurate picture of Pilo-inducedlearning and memory deficits.

Our findings corroborate and extend previous investiga-tions of KA- and Pilo- induced learning and memory deficits,however, to the best of our knowledge, this is the first reportof both NOR and NPR testing at multiple delay lengths in KAand Pilo treated animals. Other groups have reported KA-induced spatial memory deficits at delays as short as 2 and3 min on the NPR task performed once the animals are chron-ically epileptic (Chauviere et al., 2009; Gobbo and O’Mara,2004). Given that our studies were performed early afterSE when seizures are less frequent, our findings extend theliterature, showing that these deficits are present beforefrequent, chronic seizures begin. Identification of a taskthat reveals memory deficits at a time point when the like-lihood of seizure occurrence is low, allows for the studyof co-morbid cognitive impairment without the additionalconfound of persistent seizure activity.

The model- and delay-dependent effects revealed heremay help to clarify some of the discrepancies in the liter-ature regarding memory performance of chemoconvulsant-treated animals on the NOR task. For example, Chauviereet al. (2010), reported intact recognition memory in Pilo-treated rats on a variation of the NOR task, but recognitionmemory testing was performed after a 2-min delay. Ourdata would suggest a longer delay length may be requiredto reveal recognition memory deficits in the Pilo model.Another study, Detour et al. (2005) failed to find signif-icant deficits in the NOR task after a 24-h delay in thelithium-Pilocarpine model of epilepsy. Given that we foundsignificant deficits on this task at the 24-h delay in boththe KA and Pilo models, recognition memory may be differ-entially affected depending on the particular SE inductionmodel of epilepsy employed. This is not surprising given thatdifferent SE induction models can result in different pat-terns of neurodegeneration. It is possible that the perirhinalcortex, the brain region that governs recognition mem-ory, may be less affected in the lithium-Pilo model whichmay account for the observed discrepancies between themodels.

Interestingly, other studies testing performance in theopen field task have found chemoconvulsant treatmentto induce hyperactivity (Gobbo and O’Mara, 2004) anddecrease indices of anxiety (Inostroza et al., 2012). Thepresent study found locomotion and anxiety indices ofchemoconvulsant treated animals to be similar to thoseof control animals with no evidence of hyperactivity orchanges in anxiety levels. This difference in findings is likelyattributable to testing during the early phase of epilepto-genesis versus testing once chronic spontaneous seizureshave occurred. The lack of differences in locomotion and

indices of anxiety presents a unique opportunity to test forlearning and memory deficits without concomitant changesin other indices that might contribute to poor performanceon learning and memory tasks confounding interpretation.

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In conclusion, while learning and memory deficits haveeen reported in both KA and Pilo treated animals during thehronic phase of epileptogenesis, fewer reports have testedognition early after SE. The data presented here demon-trate that deficits in spatial memory are task dependent inhe Pilo model and globally impaired in the KA model. Addi-ionally, both KA and Pilo models exhibit impaired long-termecognition memory. Future studies can use this informationo study cognitively targeted therapeutics in these modelsnd should future studies chose to utilize the NOR and NPRasks, we have shown the delay lengths were deficits areresent.

cknowledgments

his work was funded by Grants NIHRO1NS039587—11MP), NIHRO1NS039587—11 S1 (J.P.), R21NS072099 (M.P.)O1NS083422 (M.P.), R21NS072099 (M.P.), and the CUREultidisciplinary award 2011 (M.P. & Roberts). The authorsould like to thank Michael Hall and the Neuroscienceachine Shop supported by the Rocky Mountain Neurologicalisorders Core Center Grant NIH/NS048154 for manufac-ure of behavioral testing arenas. We would also like tocknowledge the Center for NeuroScience Animal Behaviorore where the entirety of the studies were performed. Theuthors would also like to thank Dr. Karen Stevens for heraluable feedback on early drafts of this manuscript.

eferences

brahams, S., Morris, R.G., Polkey, C.E., Jarosz, J.M., Cox, T.C.S.,Graves, M., Pickering, A., 1999. Hippocampal involvement inspatial and working memory: a structural MRI analysis of patientswith unilateral mesial temporal lobe sclerosis. Brain Cogn. 41,39—65.

dams, S.J., O’Brien, T.J., Lloyd, J., Kilpatrick, C.J., Salzberg,M.R., Velakoulis, D., 2008. Neuropsychiatric morbidity in focalepilepsy. Br. J. Psychiatry 192, 464—469.

kanuma, N., Hara, E., Adachi, N., Hara, K., Koutroumanidis, M.,2008. Psychiatric comorbidity in adult patients with idiopathicgeneralized epilepsy. Epilepsy Behav. 13, 248—251.

en-Ari, Y., 1985. Limbic seizure and brain damage produced bykainic acid: mechanisms and relevance to human temporal lobeepilepsy. Neuroscience 14, 375—403.

rooks-Kayal, A.R., Bath, K.G., Berg, A.T., Galanopoulou, A.S.,Holmes, G.L., Jensen, F.E., Kanner, A.M., O’Brien, T.J., Whit-temore, V.H., Winawer, M.R., Patel, M., Scharfman, H.E., 2013.Issues related to symptomatic and disease-modifying treat-ments affecting cognitive and neuropsychiatric comorbidities ofepilepsy. Epilepsia 54 (4), 44—60.

hauviere, L., Rafrafi, N., Thinus-Blanc, C., Bartolomei, F.,Esclapez, M., Bernard, C., 2009. Early deficits in spatial mem-ory and theta rhythm in experimental temporal lobe epilepsy. J.Neurosci. 29, 5402—5410.

hristensen, J., Vestergaard, M., Mortensen, P.B., Sidenius, P.,Agerbo, E., 2007. Epilepsy and risk of suicide: a population-basedcase-control study. Lancet Neurol. 6, 693—698.

etour, J., Schroeder, H., Desor, D., Nehlig, A., 2005. A five monthperiod of epilepsy impairs spatial memory, decreases anxiety,but spares object recognition in the lithium-pilocarpine model

in adult rats. Epilepsia 46 (4), 499—508.

’Hooge, R., De Deyn, P.P., 2001. Applications of the Morris watermaze in the study of learning and memory. Brain Res. Rev. 36,60—90.

1

D

E

F

F

G

G

H

H

H

I

J

K

M

M

N

P

P

P

R

S

S

S

T

Tactivity in rats. Pharmacol. Biochem. Behav. 31, 959—962.

Viskontas, I.V., McAndrews, M.P., Moscovitch, M., 2000. Remote

040

ix, S.L., Aggleton, J.P., 1999. Extending the spontaneous pref-erence test of recognition: evidence of object-location andobject-context recognition. Behav. Brain Res. 99, 191—200.

lger, C.E., Helmstaedter, C., Kurthen, M., 2004. Chronic epilepsyand cognition. Lancet Neurol. 3, 663—672.

ilho, G.M., Mazetto, L., da Silva, J.M., Caboclo, L.O., Yacubian,E.M., 2011. Psychiatric comorbidity in patients with two proto-types of focal versus generalized epilepsy syndromes. Seizure20, 383—386.

rucht, M.M., Quigg, M., Schwaner, C., Fountain, N.B., 2000. Dis-tribution of seizure precipitants among epilepsy syndromes.Epilepsia 41, 1534—1539.

obbo, O.L., O’Mara, S.M., 2004. Post-treatment, but notpre-treatment, with the selective cyclooxygenase-2 inhibitorcelecoxib markedly enhances functional recovery from kainicacid-induced neurodegeneration. Neuroscience 125, 317—327.

uerreiro, C.A.M., Jones-Gotman, M., Andermann, F., Bastos, A.,Cendes, F., 2001. Severe amnesia in epilepsy: causes, anato-mopsychological considerations, and treatment. Epilepsy Behav.2, 224—246.

aut, S.R., Hall, C.B., LeValley, A.J., Lipton, R.B., 2007. Canpatients with epilepsy predict their seizures? Neurology 68,262—266.

ellier, J.L., Patrylo, P.R., Buckmaster, P.S., Dudek, F.E., 1998.Recurrent spontaneous motor seizures after repeated low-dosesystemic treatment with kainate: assessment of a rat model oftemporal lobe epilepsy. Epilepsy Res. 31, 73—84.

ermann, B., Seidenberg, M., Jones, J., 2008. The neurobe-havioural comorbidities of epilepsy: can a natural history bedeveloped? Lancet Neurol. 7, 151—160.

nostroza, M., Cid, E., Menendez de la Prida, L., Sandi, C., 2012.Different emotional disturbances in two experimental modelsof temporal lobe epilepsy in rats. PLoS ONE 7 (6), e38959,http://dx.doi.org/10.1371/journal.pone.0038959.

ett, D.A., 2010. Finding new cures for neurological disorders: apossible fringe benefit of biodefense research. Sci. Transl. Med.2, 23ps12.

ealy, J., Commins, S., 2011. The rat perirhinal cortex: a review ofanatomy, physiology, plasticity, and function. Prog. Neurobiol.

93, 522—548.

umby, D.G., 2001. Perspectives on object-recognition memory fol-lowing hippocampal damage: lessons from studies in rats. Behav.Brain Res. 127, 159—181.

J.N. Pearson et al.

umby, D.G., Gaskin, S., Glenn, M.J., Schramek, T.E., Lehmann,H., 2002. Hippocampal damage and exploratory preferences inrats: memory for objects, places, and contexts. Learn. Mem. 9,49—57.

akken, K.O., Solaas, M.H., Kjeldsen, M.J., Friis, M.L., Pellock,J.M., Corey, L.A., 2005. Which seizure-precipitating factors dopatients with epilepsy most frequently report? Epilepsy Behav.6, 85—89.

inel, J.P.J., Mana, M.J., 1989. Adaptive interactions of rats withdangerous inanimate objects: support for a cognitive theoryof defensive behaviour. In: Blanchard, R.J., Brain, P.F., Blan-chard, D.C., Parmigiani, S. (Eds.), Ethoexperimental Approachesto the Study of Behaviour. Kluwer Academic, Dordrecht,pp. 137—150.

loner, C.J., Gaymard, B.M., Rivaud-Pechoux, S., Baulac, M.,Clemenceau, S., Samson, S., Pierrot-Deseilligny, C., 2000.Lesions affecting the parahippocampal cortex yield spatial mem-ory deficits in humans. Cereb. Cortex 10, 1211—1216.

itkanen, A., Sutula, T.P., 2002. Is epilepsy a progressive disor-der? Prospects for new therapeutic approaches in temporal-lobeepilepsy. Lancet Neurol. 1, 173—181.

acine, R.J., 1972. Modification of seizure activity by electricalstimulation: II. Motor seizure. Electroencephalogr. Clin. Neuro-physiol. 32, 281—294.

arkisian, M.R., 2001. Overview of the current animal modelsfor human seizure and epileptic disorders. Epilepsy Behav. 2,201—216.

perk, G., 1994. Kainic acid seizures in the rat. Prog. Neurobiol. 42,1—32.

pector, S., Cull, C., Goldstein, L.H., 2000. Seizure precipitantsand perceived self-control of seizures in adults with poorly-controlled epilepsy. Epilepsy Res. 38, 207—216.

remblay, E., Nitecka, L., Berger, M.L., Ben-Ari, Y., 1984. Maturationof kainic acid seizure brain damage syndrome in the rat: I. Clin-ical, electrographic and metabolic observations. Neuroscience13, 1051—1072.

reit, D., Fundytus, M., 1988. Thigmotaxis as a test for anxiolytic

episodic memory deficits in patients with unilateral temporallobe epilepsy and excisions. J. Neurosci. 20, 5853—5857.