Age-dependent decline in learning and memory performances of WAG/Rij rat model of absence epilepsy

Growing number of studies started to indicate that epilepsy is not restricted to recurrent seizures and emphasize that neuropsychological symptoms are part of its clinical profile [1, 2]. Furthermore, emotional and cognitive dysfunction impacts academic performance and social life not only in drug-resistant epilepsy but also benign epileptic syndromes including absence epilepsy, a neurological disorder that lacks structural deficits and responds well to treatment [2–7]. In addition to neuropsychiatric comorbidities, recent studies investigated the time course of neurocognitive impairments in epileptic patients [8–12]. For instance, Hermann et al. reported progressive cognitive abnormalities in temporal lobe epilepsy patients over a 4-year period compared to age- and sex-matched healthy controls [9]. In another study, Hommet et al. compared adolescents and young adults in complete recovery from benign childhood epilepsy with centrotemporal spikes (BECTS) and childhood absence epilepsy (CAE) [8]. They found that patients with CAE showed decline in intellectual performance. Furthermore, brain-imaging studies demonstrated that the brain regions implicated in behavior, cognition, and language had different developmental characteristics in CAE [11, 12]. These studies overall emphasize the importance of studying age-related aspects of epilepsy for a better understanding of its multi-faced nature and the development of effective tools for tracking and preventing its progression.

In order to elucidate the relation between behavioral/cognitive impairment and epilepsy, research studies have often used models of acquired epilepsy such as kindling or status epilepticus [13–18]. These models, in which limbic structures are affected, are characterized by convulsive seizures. On the other hand, research on genetic models of absence epilepsy has primarily focused on emotional dysfunction and paid relatively less attention to cognitive impairment [19–22]. Moreover, although genetic models are particularly well-suited for detecting and tracking developmental dynamics, age-related behavioral and cognitive alterations in these models remain unexplored.

Wistar-Albino-Glaxo from Rijswijk (WAG/Rij) rats, a strain of Wistar origin, is a widely used, valid genetic model of generalized absence epilepsy [23]. Electrophysiologically and behaviorally well-defined absence seizures occur in every member of WAG/Rij rats [23]. Spike-wave complexes (SWD) begin to emerge on the epidural records of two month-old WAG/Rij rats and their frequency and duration increase with age [23]. SWD activity has been suggested to be closely related to depression-like symptoms observed in this model [19, 24]. Unlike the depression-like behavior however, the evaluation of the absence epilepsy models in terms of anxiety-like behaviors has not revealed consistent strain differences [25].

The well-documented impact of epilepsy and depression on learning and memory processes, and the existence of relatively few cognitive characterizations of animal models of absence epilepsy in the literature motivated our study. We examined learning and memory performance of 5 months and 13 months old WAG/Rij rats and age-matched Wistar controls in passive avoidance and Morris water maze, two tasks that are well-characterized in terms of their behavioral, neuroanatomical, and neurochemical bases. We further evaluated the locomotor activity and anxiety-like behavior of 5 m and 13 m WAG/Rij and age-matched Wistar rats using locomotor activity cage and elevated plus maze, respectively. Results revealed age-dependent differences between WAG/Rij and Wistar rats in emotional and spatial memory in the absence of clear differences in terms of anxiety-like behavior and locomotor activity.

WAG/Rij rats exhibited worse emotional and spatial memory performances in passive avoidance and Morris water maze tests compared to their aged-matched Wistar controls at 13 months but not at 5 months of age. Importantly, these cognitive deficits were observed in the absence of clear differences in anxiety-like behavior and locomotor activity. For instance, WAG/Rij rats did not exhibit different levels of anxiety-like behavior (i.e., time spent in open arms or the ratio of time spent in open arms to time spent in open or closed arms) in the elevated plus maze or different levels of locomotor activity in the activity cage compared to their age-matched Wistar controls. These results overall suggested an age-dependent decline in the learning and memory performances of WAG/Rij rat model of absence epilepsy (compared to Wistar controls) that cannot be accounted for by differential levels of anxiety and locomotor activity.

The idiopathic generalized epilepsy, characterized with typical absence seizures, has been assumed to be a type of benign epilepsies. However, recent studies led to a change in this assumption except for the responsiveness of these conditions to anti-epileptic treatment [11, 12, 27]. Clinical studies carried out with absence epileptic patients indicate presence of cognitive impairments including general cognitive decline [28], visiospatial dysfunction [28, 29], linguistic problems [26], non-verbal and short-term verbal memory impairment [26, 29] as well as attentional, emotional and behavioral alterations [9, 29–32]. Several of these studies have been conducted with non-medicated patients with newly emerging seizures [9, 29]. In humans, several variables including medical treatment and social factors make it difficult to detect the onset and track the progression of cognitive impairments [33].

In animal models of absence epilepsy, cognitive functions have received surprisingly little attention. In one of these studies, WAG/Rij rats were tested on the hole board and radial arm maze, and exhibited a partial decline of the reference memory without the working memory impairment [34]. Bazyan et al. ([2000]) also showed that WAG/Rij rats performed worse than controls in the passive avoidance test [35]. In the same study, although WAG/Rij rats performed better than controls early in training in the active avoidance test, their performance worsened during latter trials [35]. These results emphasize the task-specificity of memory performance in the absence epilepsy models. None of these studies however, have addressed the possible age-dependent dynamics in memory performance.

Our study clearly demonstrates that memory impairment became evident in WAG/Rij rats even over the course of a limited age range (i.e., 5 m/young adulthood vs. 13 m/middle-aged). One plausible causal basis of this age-dependent decline in memory performance of WAG/Rij rats in the light of previous studies is their differential spike-wave discharge (SWD) activity compared to age-matched Wistar rats. For instance, previous studies showed an age-dependent increase in the frequency of absence seizures in WAG/Rij rats; SWD activity starts between 2–3 months of age and reaches hundreds a day by 12 months of age, the age group tested in this study [23, 36]. SWD activity does not only appear in these inbred absence epilepsy models but also in g/Cpb rats [37], APO-SUS [38], and Long Evans [39] rats. Importantly, these rat models too exhibit impaired performance on different memory and/or emotional tasks [37, 38, 40]. SWD activity was further observed in all members of the outbreed Wistar rats within the 15–23 m age range [41] and can be detected even earlier in this strain [42]. Again consistent with SWD-based account, Wistar rats exhibit poor performance in passive avoidance and water maze tests within the older age range characterized by SWD activity [43–45]. Thus, the behavioral results of the current study combined with electrophysiological findings of previous studies point at a possible relation between SWD activity and age-dependent memory impairment of WAG/Rij rats. Note that since we did not record EEG in this study, this relation only remains to be a plausible account that needs to be substantiated by data. Thus, future studies are needed to establish the relation between SWD activity and memory performance by recording EEG from rats tested in learning and memory assays.

Despite these previous findings that suggest a potential relationship between SWD activity and cognitive dysfunction, results obtained from GAERS (genetic absence epilepsy rats from Strasbourg) appear to conflict this relation. The SWD activity emerges earlier, occurs more frequent and with longer duration in GAERS, another genetic rat model of absence epilepsy [23]. Thus, under an SWD-based account GAERS would be predicted to exhibit poorer memory performance earlier than observed here. Contrary to this prediction, GAERS rats performed better than controls in the two-way active avoidance test [46]. The disparity between the effect of brain lesions on two-way active avoidance and other memory task performances suggest that this contradiction might be task-specific.

For instance, hippocampal, septohippocampal or reticular thalamic nucleus lesions, which had detrimental effects on performances in many memory tasks, have been shown to enhance two-way active avoidance performance [47–51]. Based on this disparity, it has been suggested that two-way active avoidance performance entailed complex aversive learning, and that it could be facilitated by reduced emotionality, decreased cholinergic innervation, and increased behavioral disinhibition [50, 52]. The study of GAERS rats in other memory tests would help elucidate whether their enhanced performance in two-way active avoidance is task specific or not.

A neuroanatomical perspective would contribute to the understanding of the dynamics that underlie our behavioral results. Limbic structures including amygdala, hippocampus, and parahippocampal cortical areas are particularly implicated for the performance on the two memory tasks used in this study [53–56]. These structures were evaluated previously by electrophysiological, molecular, and functional methods in the absence epileptic rats. Electrophysiological studies have shown that limbic areas were silent during thalamocortical SWD activity [57, 58]. On the other hand, other studies reported the presence of the metabolic and molecular changes [59–62]. Support for functional changes in the limbic structures of absence epileptic rats originate from kindling studies. Kindling, which is a model of epileptogenesis for acquired epilepsies, shares some of the similar mechanisms with long-term potentiation (LTP) [57]. In fact, it is considered to be a pathological form of neuroplasticity [63, 64]. Development of kindling has been shown to be more difficult in rats with absence epilepsy and different degrees of resistance to kindling progression have been shown in these animals in amygdala, hippocampus, and perirhinal cortex [65–67]. If resistance to kindling in these rat models indeed generalizes to the formation of LTP in the corresponding limbic structures, this could predict worse learning and memory performance in absence epilepsy rat models.

Finally, the performance of rats in these tests might be altered by other factors such as emotional state, motivation and/or impulsivity. For instance, in a recent study [18] of two models of temporal lobe epilepsy in two different strains, researchers found that lower anxiety scores (as assessed by open arm related measures) in EPM was related to lower performance on the Morris water maze. Although we did not observe any differences between WAG/Rij and Wistar rats in terms of anxiety-like behavior as canonically assessed by open arm related measures, WAG/Rij rats spent less time in closed arms compared to age-matched Wistar rats at 13 months of age. This result might imply weaker safety-seeking tendencies of WAG/Rij rats and might indeed be relevant to their lower performance on passive avoidance and Morris water maze, which are tasks with aversive components. Contrary to these arguments however, van der Staay et al. ([2009]) demonstrated that emotional reactivity as assessed by open field and elevated plus maze activities did not mediate learning and memory performance in tasks with aversive components such as Morris water maze and passive avoidance [68].

In our study, WAG/Rij rats did not exhibit different levels of locomotor activity in the activity cage however they exhibited higher total number of arm entries in EPM at both ages. Although, these differences might be argued to have mediated the performance on passive avoidance and Morris water maze, this reasoning cannot fully account for the age-dependence of poorer performance on these memory tasks. In other words, differences between WAG/Rij and Wistar rats in terms of arm entries was comparable between the two age groups whereas cognitive impairment appeared specifically at 13 months of age. The same argument also holds for the possible relation between number of arm entries and time-spent in closed arms; the between-strain differences for the former measure was not age-dependent whereas age-dependency of between-strain differences held for the latter. Furthermore, although previous studies argued for a relation between higher levels of activity in elevated plus maze and open arm exploration [69], its relation to closed arm exploration has not been discussed.

We did not test rats on a cued-learning trial in order to control for possible factors (other than spatial memory) that might have mediated the observed performances. However, note that analysis of the escape latencies normalized by the escape latency of the first session also revealed similar results. We observed that WAG/Rij rats at 13 months of age jumped of the platform upon being placed on it by the experimenter after the trial cut-off duration. This behavior was not observed with any Wistar rats or 5 months old WAG/Rij rats. Further studies should evaluate the swimming speed and other potentially confounding factors.

In conclusion, this study shows an age-dependent decline in the learning and memory performance of WAG/Rij rats compared to age-matched Wistar rats in the absence of clear differences in anxiety-like behavior and locomotor activity. More detailed studies are needed to understand the electrophysiological correlates of these behavioral alterations as well as the relation between the neuroanatomical and neurochemical components of this disturbance and other emotional/behavioral comorbid situations.