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Summary: Purpose: Seizures in the developing brain cause less macroscopic structural damage than do seizures in adulthood, but accumulating evidence shows that seizures early in life can be associated with persistent behavioral and cognitive impairments. We previously showed that long-term spatial memory in the eight-arm radial-arm maze was impaired in rats that experienced a single episode of kainic acid (KA)-induced status epilepticus during early development (postnatal days (P) 1–14). Here we extend those findings by using a set of behavioral paradigms that are sensitive to additional aspects of learning and behavior.
Methods: On P1, P7, P14, or P24, rats underwent status epilepticus induced by intraperitoneal injections of age-specific doses of KA. In adulthood (P90–P100), the behavioral performance of these rats was compared with that of control rats that did not receive KA. A modified version of the radial-arm maze was used to assess short-term spatial memory; the Morris water maze was used to evaluate long-term spatial memory and retrieval; and the elevated plus maze was used to determine anxiety.
Results: Compared with controls, rats with KA seizures at each tested age had impaired short-term spatial memory in the radial-arm maze (longer latency to criterion and more reference errors), deficient long-term spatial learning and retrieval in the water maze (longer escape latencies and memory for platform location), and a greater degree of anxiety in the elevated plus maze (greater time spent in open arms).
Conclusions: These findings provide additional support for the concept that seizures early in life may be followed by life-long impairment of certain cognitive and behavioral functions. These results may have clinical implications, favoring early and aggressive control of seizures during development.
Epilepsy is a common neurologic disorder that occurs much more frequently in children than in adults (1). Many data support the idea that prolonged or frequent seizures in young animals and patients lead to later cognitive deficits, which can often be subtle [e.g., (2–11)]. In both laboratory animals and humans, the brain's susceptibility to seizures is increased during specific developmental epochs, yet the behavioral consequences of seizures tend to be less pronounced when seizures occur early in life than in adulthood. Reasons for these age dependencies are not fully understood but are clearly complex (12–14).
To investigate the long-term consequences of epilepsy early in life, several animal models have been developed (3,6,7,10,13,15). Most of these models use chemoconvulsants to induce acute seizures, mimicking status epilepticus. Later, a variety of tests assessing behavioral and cognitive function, seizure susceptibility, and histologic evidence of neuronal damage is applied. For example, kainic acid (KA) has been used to investigate the behavioral consequences of status epilepticus at various ages. In adult animals, KA causes an epilepsy syndrome similar to human temporal lobe epilepsy, with mesial temporal sclerosis, spontaneous seizures, and significant deficits in learning and memory (16). In developing animals compared with adults, however, the long-term effects of KA-induced status epilepticus are less severe in terms of macroscopic structural damage and behavioral impairments (17–21). When administered intraperitoneally at ages younger than about postnatal day (P) 20 in rats, KA does not cause significant hippocampal cell loss or recurrent seizures, and synaptic reorganization (mossy fiber sprouting) is less pronounced than that in adults, even if the seizures are more severe in the pups (19). These observations are not restricted to the KA model; seizures induced by kindling, pilocarpine, and flurothyl are likewise not associated with significant synaptic reorganization, as assessed by mossy fiber sprouting (22,23). Nevertheless, seizure-induced behavioral impairments may occur in later life in response to status epilepticus during early development (24–26).
Depending on the method of seizure induction, dose, age of administration, and behavioral testing, investigators have found varied effects of early seizures on subsequent learning and behavior (25–29). We recently provided additional evidence for long-term adverse cognitive changes after KA-induced status epilepticus in early development (30). KA seizures were evoked during early postnatal development (P1–P24). A seizure between P1 and P14 was associated with life-long impairment in spatial learning and memory in the radial-arm maze. These rats also exhibited impaired induction of hippocampal long-term potentiation (LTP). Seizure-induced alteration of synaptic plasticity also was manifested as a long-term reduction in susceptibility to kindled seizure development and enhanced paired-pulse inhibition in the dentate gyrus (30). Such chronic behavioral effects of early seizures on hippocampal-based memory demonstrate the influence of activity-dependent and seizure-induced plasticity during development on subsequent cognitive function in adulthood. Together, these findings suggest that seizures early in development are not as harmless as was once thought (24).
The present study addressed the behavioral effects of KA-induced status epilepticus at various stages of hippocampal development (31), focusing on whether early-life seizures differentially alter several hippocampal-dependent behavioral and cognitive processes, including short- and long-term spatial learning and memory, anxiety, and exploratory activity.
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- MATERIALS AND METHODS
In KA-treated rats of all ages, status epilepticus developed, with a sequence of clinical features that differed somewhat by age (20,30). P1 and P7 rats initially became immobile with loss of limb tone and ataxia. They then exhibited intermittent hyperactivity with rhythmic “bicycling” movements of all extremities, opisthotonic arching, and tonic limb extension. Previous studies with implanted hippocampal electrodes showed that such clinical signs represent electrographic ictal activity (30). In older rats (P14, P24), KA-induced seizures consisted of initial immobility and staring. At P14, repetitive scratching movements and rudimentary wet dog shakes (WDSs) were seen. At P24, WDSs were prominent, followed by facial clonus, masticatory automatisms, and increased salivation; tonic–clonic movements of one or more limbs, rearing, and falling then ensued. At all ages, seizures (status epilepticus) persisted intermittently for 2 to 6 h. The status epilepticus was not terminated pharmacologically. Later, spontaneous seizures were not monitored specifically, but none was observed during routine handling and behavioral testing.
The performance of rats in the radial-arm maze was judged by the latency to find and consume the four baits, the total number of arm entries, and the number of arm entry “errors.” Entering an arm without bait present is referred to as a reference memory error, whereas entering an arm in which the bait had already been consumed is referred to as a working memory error. The number of total arm entries is a reflection of anxiety; a more anxious rat would tend to remain stationary and explore the maze less (fewer arm entries). Therefore each of these criteria measures a different aspect of radial-arm maze performance.
All groups of rats learned to find and consume the four baits in each trial (Fig. 1). As a function of trial number, the latency to reach criterion (find and eat all four baits) was significantly longer in rats that received KA (Fig. 1). By post hoc testing, differences were significant when comparing the first trial with the fifth and sixth trials, for each age group. It took significantly more time for the KA-treated P1, P7, P14, and P24 rats to find the baits than for the controls.
Figure 1. Radial-arm maze performance: latencies to criterion. Rats underwent kainic acid (KA)–induced status epilepticus at various postnatal ages [P1 (A), P7 (B), P14 (C), P24 (D)] and were tested in the radial-arm maze as adults (P90–P100). The age-matched controls received saline rather than KA. Testing consisted of six trials on a single day. The latency to criterion (time to find four baits) is plotted against trial number. In each age group, significant differences become apparent between experimentals and controls by the fifth trials (*). See text and Table 1. In each plot, solid circles are controls and open triangles are experimentals (KA-treated).
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In addition, in each age group, fewer reference errors (entries into unbaited arms) were committed over time (i.e., with consecutive trials; Fig. 2). Post hoc analysis showed that, for each age group, by the third or fourth trial, a significant difference existed between controls and experimentals in terms of the total number of reference errors, with the animals that experienced seizures committing more errors (Fig. 2).
Figure 2. Radial-arm maze performance: total number of reference errors (entries into unbaited arms). Rats underwent kainic acid (KA)–induced status epilepticus at various postnatal ages [P1 (A), P7 (B), P14 (C), P24 (D)] and were tested in the radial-arm maze as adults (P90–P100). The age-matched controls received saline rather than KA. Testing consisted of six trials on a single day. The number of reference errors is plotted against trial number. Significantly more reference errors were committed by the treated animals on the third through sixth trials at ages P1 (A), P7 (B), and P14 (C), and on the fourth through sixth trials at P24 (D). See text and Table 1. In each plot, solid circles are controls and open triangles are experimentals (KA-treated).
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Previous studies using the radial-arm maze showed that anxious rats are less active and avoid exploring arms of the maze (38,39). Therefore to rule out an effect of anxiety on radial-arm maze performance between control and experimental groups, the total number of arm entries (baited, unbaited, previously baited) was compared (Fig. 3). On the first trial, no difference was found between controls and any KA-treated group as to the total number of arm entries (p < 0.05; Fig. 3, First Trial). Therefore anxiety was not a factor limiting initial arm exploration. By the sixth trial, controls and each KA-treated group made significantly fewer total arm entries compared with the first trial (i.e., they each learned the task, as described earlier). However, on the sixth trial, the control animals had significantly fewer total arm entries than each KA-treated group (p < 0.05; Fig. 3, Sixth Trial). These findings suggest that differences in total arm entries from the first trial to the sixth trial cannot be explained by a difference in anxiety between the groups.
Figure 3. Radial-arm maze performance: total number of entries into baited, unbaited, and previously baited arms. The total number of arm entries is shown for the first and sixth trials in controls and all KA-treated age groups. The number of arm entries is proportional to the level of anxiety. On the sixth trial, control rats and each age group of rats treated with KA had significantly fewer arm entries compared with the first trial (*); these data indicate that all rats learned the task and required fewer entries as a function of trial number. On the sixth trial, each KA-treated group had significantly more arm entries than controls (**); these data indicate that anxiety differences cannot explain radial-arm maze learning differences between control and seizure groups.
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During the habituation trial, the rats swam randomly around the pool, with no preference for a particular quadrant. The acquisition of place learning is shown in Fig. 4A, in which escape latencies are plotted against day of training. All rats learned to find the hidden platform and escape onto it, as indicated by the progressive decrease in mean escape latency over the 4 training days in each group. The escape latencies for the experimental groups that received KA on P1, P7, or P24 differed significantly from untreated controls (by experimental groups: F= 14.9, p < 0.0001; by days of testing, F= 42.0, p < 0.0001; Group × Days interaction, F= 4.5, p = 0.0009). By post hoc testing, the performance of the P7 and P24 KA-treated groups differed significantly from controls, whereas P1 KA-treated rats performed similar to controls. Although the performance of the rats that received KA on P7 reached control values by testing day 4, their overall learning was much slower than controls, as evidenced by results on testing days 1, 2, and 3, each of which differed significantly from controls on post hoc testing.
Figure 4. Water maze testing. A: Acquisition learning of platform location, plotting escape latency versus day of testing for controls (filled circles) and rats treated with kainic acid (KA) at various postnatal ages (P1, open circles; P7, filled triangles; P24, open triangles). All groups learned to find and escape onto the platform over the four testing days, but learning was significantly slower in KA-treated rats. B: Probe test. After the final acquisition trial, the platform was removed, and the rats were placed back in the pool for 60 s. The distance (proportional to time) swum in each quadrant was calculated. Compared with control rats, each KA-treated group spent significantly less time in the target quadrant (*), where the platform was previously located. C: Probe test. The percentage distance in each quadrant is shown for controls and rats treated with KA on P24. Control rats swam significantly longer in the target quadrant, whereas P24-treated rats spent similar time in each of the four quadrants. Similar results were found for the P1 and P7 groups (data not shown).
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On the probe test (Fig. 4B), the distance spent swimming in the target quadrant (proportional to the dwell time spent in that quadrant) differed between controls and each experimental age group (P1, P7, P24; F= 6.4; p < 0.002 by one-way ANOVA with post hoc Student–Newman–Keuls test). In Fig. 4C, the probe test results for the P24 KA group is compared with those of controls. For controls, the distance swum in the target quadrant differed from that in each other quadrant (F= 26.0; p < 0.0001), whereas for rats treated with KA on P24, swimming distances did not differ between quadrants (F= 0.97; p = 0.42). Similar results were found with the P1 and P7 groups (data not shown). Therefore each KA-treated group showed deficits in spatial bias by spending equivalent time in all quadrants rather than dwelling in the target quadrant.
Elevated plus maze
Anxiety scores were lower (indicating greater anxiety levels) in all age groups (P1, P7, P14, P24) among rats that had experienced KA–induced status epilepticus compared with the controls that did not receive KA (Fig. 5A; F= 26.7; p < 0.0001; post hoc Bonferroni test, p < 0.05). These findings demonstrate that increased anxiety existed in all experimental groups, and they spent less time in the open arms, compared with the controls.
Figure 5. Elevated plus maze and open-field testing. A: Anxiety scores (time spent in open arms as a proportion of total maze time) is plotted for controls and rats treated with kainic acid (KA) at various ages (P1–P24). All KA-treated groups were significantly more anxious than were controls. The number of rats (n) in each group is indicated below each bar. B: Exploratory behavior in the open field. Number of head dips into holes in the floor of the open field box is plotted for controls and rats treated with KA at various ages (P1–P24). Only the P24-treated group differed from controls. C: Locomotor activity in the open field. The number of squares visited (open bars) and number of rears (filled bars) are plotted for controls and rats treated with KA at various ages (P1–P24). None of the KA-treated groups differed from controls. The results in B and C show that the anxiety differences seen in A were not due to inherent differences in locomotor activity.
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On open-field testing, no difference was seen in exploratory activity (number of head dippings) or locomotor activity (number of squares visited and rearings) between age groups or between controls and rats that had experienced KA seizures (Fig. 5B and C). Therefore the differences in anxiety scores between the control and experimental groups were not due to an alteration of locomotor or exploratory activity and may therefore be a consequence associated with the seizure history.
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These results add to the accumulating evidence that seizures during early brain development can result in life-long behavioral and cognitive deficits. In this study, status epilepticus induced by the glutamate receptor agonist KA early in life was associated with significant learning and memory deficits and persistent anxiety in adulthood. As summarized in Table 1, KA seizures on P1, P7, P14, and P24 (radial-arm maze) or P7 and P24 (water maze) were associated with both short- and long-term impairments in spatial learning and memory, respectively, both of which are hippocampus-dependent functions. The finding that controls and rats that received KA on P1 did not differ on water maze testing, whereas P1 rats demonstrated impaired radial-arm maze learning, suggests that the latter test may be more sensitive for seizure-induced behavioral dysfunction. Furthermore, rats that experienced status epilepticus at these early ages exhibited heightened levels of anxiety as adults (elevated plus maze), again correlating with abnormal hippocampal/limbic system function. The lack of major hippocampal structural damage in other studies using similar protocols (18,19,30) suggests that seizures early in life can have long-term cognitive consequences despite the absence of overt cellular damage. Although some synaptic reorganization may occur after multiple postnatal seizures (40), its functional consequences are unclear and may differ by age. It is already known that KA–induced status epilepticus in adult rats causes a marked disruption of spatial learning and memory, far greater than the changes seen in developing rats (21,41).
Table 1. Summary of results
|Radial-arm maze||Short-term spatial learning and memory||Animals with KA seizures early in life (P1, P7, P14, P24): – took longer to learn food location – made more reference errors (entries into unbaited arms) – no difference was found in performance as a function of age at which seizure occurred.|
|Water maze||Long-term spatial learning and memory||Animals with KA seizures early in life (P1, P7, P24): – took longer to learn platform location (except P1) – spent less time swimming in target quadrant (probe test) – deficits varied inversely with age at the time of status epilepticus: P24 > P7 > P1.|
|Elevated plus maze||Anxiety||Animals with KA seizures early in life (P1, P7, P14, P24):- – were more anxious (preferred closed arms).|
|Open-field test||Locomotion, exploration||Animals with KA seizures early in life (P1, P7, P14, P24): – exhibited no difference from controls in head dips, squares visited, or number of rears (therefore anxiety differences were not explainable on basis of differences in locomotion).|
These experiments confirm and extend those of Lynch et al. (30). In that study, a different version of the radial-arm maze was used, with the protocol requiring that rats learn the location of the food pellet over several days of testing, until a criterion was reached (consuming all food pellets in the first five arm entries). The task was considered “learned” once the animal achieved five consecutive criterion performances. Here, we used a modified radial-arm maze protocol, in which all learning and testing occurred over multiple trials on a single day. In this abbreviated training condition as well, KA seizures early in development were associated with impaired learning of the task and more reference errors compared with those in controls. However, compared with the previous data (30), our results in the radial-arm maze had no relation to age. That is, the latency to criterion, number of reference errors, and total errors were similar, regardless of the age at which status epilepticus occurred. Similarly, in the elevated plus maze, anxiety levels were increased to a similar degree in each KA-treated group. Therefore the fact that status epilepticus occurred had a greater impact than the age at which it occurred.
In this study, we used an additional measure of hippocampal integrity, the water maze, a well-validated test of spatial learning and memory (33,42,43). In this task, rats that were treated with KA on P7 or P24 learned the platform location slower than did controls, with a rank order of performance (worst to best): P24 > P7 > P1, controls. Therefore performance varied inversely with the age at which KA status epilepticus occurred. On the probe (spatial bias) component of the water maze, rats are allowed to swim freely without a platform present, to assess their preference (memory) for platform location in the target quadrant. Each KA-treated group (P1, P7, P24) spent significantly less time swimming in the target quadrant than controls, suggesting that the seizure groups had poorer memory of platform location.
Compared with previous studies using the Morris water maze (21), the current study detected more evidence for memory impairment as a consequence of seizures at P5, P10, and P20. The reasons for these discrepancies are uncertain, but accumulating evidence indicates that behavioral and learning deficits occur as a consequence of early-life status epilepticus. The persistence of increased anxiety from early-life status epilepticus suggests that affect is influenced as well as cognition.
Prolonged seizures caused by other etiologies [e.g., corticotropin-releasing hormone (44)] early in development also are associated with later impairment of cognitive function such as water-maze learning, and the deficits appear to be progressive over time (6). Lithium-pilocarpine status epilepticus at P16 or P20, but not at P12, was associated with cognitive impairment in the water maze in early adulthood (P55) (11).
In addition to prolonged seizures early in life leading to later cognitive abnormalities, even brief, recurrent seizures at young ages also can be detrimental (4,8,45,46). Recurrent pentylenetetrazol (PTZ) seizures in early development (P10–P14) caused significant spatial deficits in the water maze when the animals were tested on P35 and P60 (10).
The choice of model and assessment tool is critical, because different results have been obtained with different methods. The limitations of rodents as an experimental model for epilepsy were discussed recently (47,48). Furthermore, detailed knowledge of the chosen behavioral test is crucial before attributing a cognitive deficit to an experimental paradigm (25,42,43,49).
In summary, on multiple measures of hippocampus-based cognitive function, rats that experienced KA seizures during early development had persistent deficits as adults: radial-arm maze, long-term version (30), radial-arm maze, abbreviated version (present study); and water-maze acquisition learning and spatial memory (present study). These results suggest that seizures disrupt some aspect of hippocampal function during the early, vulnerable “critical period” of brain development, with deficits observable long afterward (in adulthood). Early-life KA seizures reduced long-term potentiation, increased paired-pulse inhibition in the dentate gyrus, and reduced the susceptibility to kindled seizures as adults (30). The increased inhibition was thought to be related to the loss of plasticity caused by the excessive neuronal activity of the seizure. Therefore the dentate gyrus appears to be a site of long-term cellular alterations induced by abnormal patterns of postnatal neural activity. It is likely that other hippocampal and extrahippocampal areas also are involved in these maladaptive plastic changes after early-life seizures. Indeed, each of the behavioral tests we used reflected more than just hippocampal function; rather, each also requires integration of several neural systems (31,50,51).
Together, these studies demonstrate that disruption of normal neural activity by seizures during early postnatal development produces deficits in a variety of cognitive measures and behaviors in adulthood. As such damage occurs even before the full maturation of hippocampal circuits (52), it is apparent that subsequent developmental events are not sufficient to overcome the adverse effects of early postnatal seizures. As substantial evidence indicates that status epilepticus in the developing brain does not produce the overt macroscopic damage seen in adults, the observation of long-term behavioral and memory deficits in these experiments supports the view that the term seizure-induceddamage also should include adverse functional consequences. These observations may have clinical implications for cognitive and memory dysfunction associated with epilepsy during development.