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Effect of prenatal pentylenetetrazol-induced kindling on learning and memory of male offspring
Neuroscience 172 (2011) 205–211
EFFECT OF PRENATAL PENTYLENETETRAZOL-INDUCED KINDLING ON LEARNING AND MEMORY OF MALE OFFSPRING A. POURMOTABBED,a S. E. NEDAEI,a,b M. CHERAGHI,a S. MORADIAN,a A. TOUHIDI,a M. AEINFAR,a Z. SEYFIa AND T. POURMOTABBEDc*
the other organs (Stumpf and Frost, 1978; Hallak et al., 1999; Lajoie and Mosche, 2004). Most children born to women with epilepsy are normal, but there is an increased risk of abnormal functional neurodevelopment in these children. Two to six percent of children born to an epileptic mother have a developmental delay (Koch et al., 1999) and have 2–7 fold higher rates of mental retardation than controls (Speidel and Meadow, 1972). Recent studies also indicate that generalized tonic– clonic seizures might result in cognitive problems for the child later in life, a phenomenon that is supported by evidence of significantly decreased verbal IQ scores in children whose mothers had more frequent generalized tonic– clonic seizures during pregnancy (Adab et al., 2004). Although, it has been suggested that maternal epilepsy may alter the morphology of the hippocampus of newborns, leading to cognitive impairment (Baka et al., 2004), the effects of epileptic activity on different areas on the brain have not been clearly established (Sederberg-Olsen and Olsen, 1983; Schneiderman et al., 1994). Clinical studies in this area are often difficult to interpret and compare due to exposure of fetus to antiepileptic drugs, possible influence of socio-economical factors, and parental educational level on the outcome of exposed children. Experimental animal studies can be useful in investigating the effects of seizures on neurodevelopment and in identifying the potential mechanisms involved. A periodic systemic injection of a convulsant drug, such as pentylenetetrazol (PTZ) (Assouline et al., 1984) has been shown to induce seizures in animal (Kilbey et al., 1979). PTZ is a noncompetitive antagonist of GABAA receptors. PTZ induced kindling is an accepted animal model for the study of epilepsy and its consequences on memory (Chen et al., 2003). Lamberty and Klitgaard (2000) have reported that PTZ-induced kindling disrupts spatial memory. It has been shown that kindling induced seizures cause hippocampal atrophy and neuronal loss in the limbic area (Cavazos and Sutula, 1990; Pitkanen et al., 1998), leading to learning deficits and cognitive impairment in the animal (Hamm et al., 1995; Gilbert et al., 2000; Mortazavi et al., 2005; Omrani et al., 2007), the effect of maternal seizure on the cognitive performance of newborns is, however, unknown. In this report we examined the effects of prenatal exposure to the maternal tonic-clonic seizure on the learning and memory of adolescents and adult male rats. We found, for the first time, that offspring born to epileptic mothers have significantly impaired spatial memory and passive avoidance learning compared to those born to healthy mother. These findings, thus, provide a mechanistic link
a Department of Physiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Islamic Republic of Iran b Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Islamic Republic of Iran c Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN, USA
Abstract—Current data concerning the effects of maternal seizure during pregnancy on newborns are limited. This study was carried out to investigate the effect of prenatal pentylenetetrazol (PTZ)-induced kindling on learning and memory of offspring. Female Wistar rats were kindled with i.p. injections of 25 mg/kg of PTZ on day 13 of their pregnancy. The spatial performance and passive avoidance learning of pups were tested at 7 weeks and 12 weeks of age using Morris water maze (MWM) task and shuttle-box apparatus, respectively. We found, for the first time, that prenatal exposure to maternal seizure induced by PTZ leads to a significant impairment of learning and memory. In addition, the number of live birth was significantly lower in kindled rats compared to control. In MWM studies, the young offspring of kindled rats had poor spatial learning ability. The frequent tonicclonic seizures in pregnancy was also associated with a poor memory as evidenced by decrease in distance swam in the target quadrant by the offspring of the kindled mother in the adulthood. Data obtained from shuttle-box studies showed that retention latencies of pups born to kindled dams were significantly reduced compared to those born to control dams. The hippocampus, amygdala and frontal cortex are very important for memory consolidation and our data suggest that subsequent developmental events are not sufficient to overcome the adverse effects of prenatal exposure to maternal seizures to these regions of the brain. These observations may have clinical implications for cognitive and memory dysfunction associated with epilepsy during pregnancy. Published by Elsevier Ltd on behalf of IBRO. Key words: prenatal seizure, pentylenetetrazol, Morris water maze, shuttle-box, learning and memory.
Epilepsy is one of the most common chronic diseases affecting over 1 million woman of child bearing age in the USA (Anderson et al., 1999; Bittigau et al., 2003). The infants of epileptic mothers are in higher risk for a variety of adverse pregnancy outcomes. Generalized tonic-clonic seizures increase the risk for hypoxia and acidosis, which can lead to irreversible damage to the CNS as well as to *Corresponding author. Tel: ⫹1-901-4484361; fax: ⫹1-901-4487360. E-mail address: [email protected] (T. Pourmotabbed). Abbreviations: ANOVA, analysis of variance; MWM, Morris water maze; PTZ, pentylenetetrazol. 0306-4522/11 $ - see front matter. Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2010.11.001
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between maternal epilepsy and prenatal brain development and reveal a new aspect of PTZ modulation on learning and memory.
EXPERIMENTAL PROCEDURES In vivo experiments All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of the Laboratory Animals and approved by a local Animal Ethics Committee. All efforts were made to minimize the number of animals used and their suffering. Male and female Wistar rats (Razi Institute, Tehran, Islamic Republic of Iran) aged 3– 4 months and weighting 250 –300 g at the beginning of the experiments were used. The rats were kept in animal facility room (20 –22 °C) on the 12-h light/dark cycle (lights on at 07:00 AM). Animals were housed singly in 24⫻24⫻45 cm3 transparent, plastic cage, with food and water provided ad libitum. Female rats were randomly paired with males one day after delivery to the animal care facility. Vaginal smears were performed on the females every morning (07:00 – 08:00 AM) to look for the presence of sperm. The presence of sperm marked day 1 of pregnancy (Edwards et al., 2002). Each pregnant rat was transported to her home cage and stored singly.
Kindling To test the impact of prenatal seizure on memory, on embryonic day 13 (E13) (E0 being the day in which positive vaginal smear was observed) (Baka et al., 2004), the pregnant dams were divided into two groups consisting of 10 animals each. Animals in the first group were kindled by i.p. injections of 25 mg/kg PTZ (sigma, St. Louis, MO, USA), 1 ml/kg body-weight every 15 min until seizures occurred. The total dosage of PTZ, however, did not exceed 75 mg/kg (three injections) (Klioueva et al., 2001). Pilot study showed that this dose of PTZ injected every 15 min is optimal for kindling. Immediately after injection, seizure activity was observed for 45 min and scored according to Racine (1972), modified by Becker et al. (1992), as follows: stage 0, no response; stage 1, ear and facial twitching; stage 2, myoclonic jerks without rearing; stage 3, myoclonic jerks, rearing; stage 4, turn over into side position, clonic-tonic seizures; stage 5, turn over into back position, generalized clonic-tonic seizures. Animals were considered to be kindled after reaching stage 4 or 5 seizures. The animals in the second group (the control group) received an equivalent volume of normal saline on a similar schedule. After parturition, pups were counted for each litter and weighted. Pups were then housed with their littermates until weaning at postnatal day 21 (Huang et al., 2002). Male pups from each litter were stored singly in a cage. To reduce possible litter effects, a total of four male pups from any litter were used in this experiment. Animals in each group, that is, kindled (K) and the control (C) groups were then divided into two subgroups of 16 males each according to Ehman et al. (2007). The first subgroups were allowed to grow to ⬃7 weeks (K7 and C7; adolescents) and the second subgroups were allowed to grow to 12 weeks of age (K12 and C12; adult). Within each of these subgroups, half of the rats were used for testing in the Morris water maze (MWM), while the other half was tested in the shuttle-box apparatus.
Behavioral assessment Morris water maze (MWM) apparatus. The MWM consisted of a dark circular pool of 140 cm in diameter and 70 cm in height. The pool was filled to a height of 35 cm of water, 22⫾2 °C. A transparent Plexiglas escape platform (10 cm in diameter) was located below the water surface. Pilot experiment showed that platform was not invisible to the rats. The apparatus was located
in a room with numerous extramaze cues that remained constant throughout the experiment. The distance swum to the platform (swim length), and the distance spent in each quadrant was recorded by a video tracking system. Procedure. The MWM procedure was done according to Omrani et al. (2007). Briefly, on the first day, rats were placed on the escape platform for 60 s, which was at the center of the empty pool and on the second day the rats were placed again on the platform in the same position with the pool being full of water. When the rat climbed off the platform, it was guided back on to the platform. Training started the following day. During this period, the escape platform was located in the center of the northwest quadrant and all rats were given a daily session of four trials for six consecutive days. On each trial the rat was placed in the water facing the pool wall at one of four randomly determined starting locations (north, west, east or south poles). Once the rat located the platform, it was allowed to stay on it for 30 s. If the rat was not able to find the platform within 60 s, it was guided to it and allowed to remain on it for 30 s. The rat was then returned to its heated cage for a 30 s inter-trial interval. Twenty-four hours after last training trial, spatial memory was examined in a probe trial. During this trial, the platform was removed from the pool and the rats were allowed to swim freely for 60 s. The distance spent swimming in the quadrant where the platform was previously located was recorded. To assess whether any motivational factors interfered with the rat’s ability to escape, 24 h after probe test, a visible platform trial was designed in which escape could be guided by proximal rather than distal spatial cues (Omrani et al., 2007). During this trial, the platform was raised above the water surface and placed in the southeast quadrant and extra maze cues were removed from the walls and the rats were allowed to swim freely for 60 s. The distance to platform (swim length) was recorded (Gilbert et al., 2000). Passive avoidance apparatus. The passive avoidance apparatus consisted of two light (Plexiglas) and dark (Black) compartments of the same size (20⫻20⫻30 cm3) separated by a door. The floor of the dark compartment was made of stainless-steel bars (0.5 cm diameter) separated by a distance of 1 cm. Intermittent electric shocks (50 Hz, 3 s), 1 mA intensity, were delivered to the floor of the dark compartment by an isolated stimulator (JafariSabet, 2006). Inhibitory-avoidance training. The rats were allowed to become familiar with the laboratory environment 1 h before each of the training or testing sessions. All training and testing were carried out between 08:00 AM and 12:00 AM. Each animal was placed in the light compartment for 20 s, after which the door was raised and the time the animal waited before crossing to the dark (shock) compartment was recorded as the latency. The animal was removed from the experiment when it waited for more than 180 s to cross to the other side. Once the animal completely crossed to the next compartment, the door was closed and a 1 mA foot shock was delivered for 3 s. The rat was then removed from the apparatus and 2 min later, the procedure was repeated. Training was terminated when the rat remained in the light compartment for 120 consecutive seconds. All the animals were trained with a maximum of two trials (Jafari-Sabet, 2006). Retention test. Twenty-four hours after training, a retention test was performed to examine long-term memory. Each animal was placed in the light compartment for 20 s, the door was opened, and the latency for entering into the shock compartment (as described in the training session) was measured as step through latency (STL). During these sessions, no electric shock was applied and the test session ended when the animal entered the shock compartment or remained in the light compartment for 600 s (criterion for retention) (Jafari-Sabet, 2006).
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Statistical analysis All data are presented as mean⫾SEM. Significance of the distance to platform (swim length) in MWM were assessed by twoway analysis of variance (ANOVA) with repeated measures and followed by Tukey’s test for multiple comparisons. Values for the probe trial were compared by one-way ANOVA and followed by Tukey’s test for multiple comparisons. Significance of the mean of the STL in the shuttle box was determined by the Student t-test. Differences were considered significant at the level of P⬍0.05 for all tests.
RESULTS Control animals that received saline instead of PTZ never experienced seizure activity. The female rats that were subjected to PTZ developed the characteristic features of seizure, whose severity reached stage 4/5 of kindling. PTZ-induced kindling significantly affected the number of live birth in kindled animals. Kindled rats gave birth to 9⫾0.53 (mean⫾SD) pups compared to 11⫾0.92 (mean⫾SD) pups born to control animals. This difference was statistically significant (P⬍0.001). However, there was no significant difference in pup’s body weight. The average weight of pups born to control and kindled rats were 6.6⫾0.1 g and 6.4⫾0.2 g, respectively. Learning performance in the MWM
Fig. 1. The effect of prenatal PTZ-induced kindling on spatial performance of male rats at adolescence and adulthood. Mean⫾SEM distance traveled to the platform in the water maze for each of the consecutive 6 d of training for 7-week-old (c7, n⫽8; k7, n⫽8) (A) and 12-week-old (c12, n⫽8; k12, n⫽8) (B) animals. At adolescence, significant impairment occurred only on the 5th day (* P⬍0.05). (C, D) Probe trial at adolescence and in adulthood. Each graph shows the mean percentage (⫾ SEM) of total distance swum in each quadrant for controls (c7 and c12) and kindled (k7 and k12) groups.# P⬍0.05 and
The MWM was used to assess the spatial performance and, as shown in Fig. 1, both 7-week and 12-week old rats learned to find the hidden platform, and escape onto it. Distances swum to the platform decreased over the six testing days in all experimental groups (c7, F5,35⫽9.27, P⬍0.001; c12, F5,35⫽11.74, P⬍0.001; k7, F5,35⫽3.78, P⬍0.01; and k12, F5,35⫽2.4, P⬍0.05). A two-way repeated ANOVA applied to swim lengths of 7-week-old rats found a significant group (F1,14⫽9.49, P⬍0.01) and days (F5,70⫽10.09, P⬍0.001) effects, but no groups and days interaction (F5,70⫽0.22, P⫽0.95). Post hoc comparison showed significant difference only on the 5th day (P⬍0.05) (Fig. 1A). When we tested the spatial performance of 12-week-old animals, we found a trend toward increased swim length in the k12 group of rats (group effect, F1,14⫽3.28, P⫽0.09; days effect, F5,70⫽8.09, P⬍0.001; interaction of group and days, F5,70⫽0.18, P⫽0.96) (Fig. 1B). In our experiments, spatial memory formation was measured by the percentage of distance swum in the target quadrant during probe trial. The probe test result for the k7 group is compared with that of c7 group in Fig. 1C. The distance swum by the both groups in the target quadrant differed from that of the other quadrants (c7, F3,28⫽4.4, P⬍0.05; k7, F3,28⫽5.9, P⬍0.01). The rats that were exposed to maternal seizure also demonstrated a bias for the target quadrant during the probe trial similar to that of the control group (P⬎0.05). When the performance of 12-week-old animals were tested, we found that rats in
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P⬍0.0001 for each quadrant vs. target quadrant. $ P⬍0.05 for target quadrant of k12 group vs. that of c12. The dashed line refers to chance level (25%).
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Fig. 2. The effect of prenatal PTZ-induced kindling on the passive avoidance learning. Mean⫾SEM step through latency significantly reduced in kindled groups compared to the controls either in 7-weekold (c7, n⫽8; k7, n⫽8) (A) or 12-week-old (c12, n⫽8; k12, n⫽8) (B) animals. * P⬍0.05 for kindled groups vs. controls.
the both groups swam statistically more distance in the target quadrant than in the other quadrants (c12, F3,28⫽11.97, P⬍0.0001; K12, F3,28⫽5.13, P⬍0.01). On the other hand, the distance swum in the target quadrant by the animals exposed to maternal seizure (kindled group) was significantly shorter compared with control group (P⬍0.05) (Fig. 1D). However, comparing performance of different groups on a visible platform trial showed no significant differences in swim lengths (P⬎0.05) (data not shown). Passive avoidance The step-through passive avoidance test was also used to evaluate learning and memory. Fig. 2 shows the effects of exposure to prenatal PTZ-induced kindling on memory retention of passive avoidance learning at adolescence (A) and adulthood (B). The data indicate that retention latencies of pups born to the kindled dams were significantly reduced compared to those born to control dams (P⬍0.05).
DISCUSSION Most epileptic woman can become pregnant; however, their pregnancies are prone to more complications with a
high risk of adverse effects on the fetal brain and development (Lajoie and Mosche, 2004). To date, very few well clinical trials or animal studies have addressed these issues. The present study clearly demonstrates that the exposure of the rat fetus to maternal PTZ-induced seizure has deleterious consequences on the learning and memory as indicated by water-maze and passive-avoidance tests. In the current study, pregnant animals received maximum three repetitive PTZ injections on day 13 of gestation. This stage of pregnancy was chosen for induction of kindling to specifically assess the effect of any potential maternal seizure-induced alteration of the brain construction associated with defects in neurogenesis or neuronal migration on memory of the offspring. According to Hatten (1993, 1999) neuronal proliferation in pups is continuing at this time and the epileptic seizures experienced on the 13th day of pregnancy result in migration failure of hippocampal neurons (Baka et al., 2004). In the present study, we have found that the number of live birth was significantly reduced in the kindled compared to control dams. This is consistent with the idea presented by Morrell and Montouris (2004), that seizures may lead to stimulation or inhibition of the hypothalamus, depending on the region affected by seizure and hence on endocrine disorders such as hypothalamic-pituitary-gonadal axis, leading to a reduced number of live births. There is some evidence that epileptic women give birth to children with reduced birth weight (for review see Hvas et al., 2000). In our study, however, there was no difference in body weight of the pups born to the kindled dams compared to those born to the control dams. Thus, gross intrauterine development did not seem to be influenced by whether the dam was epileptic or not as previously reported (Raffo et al., 2009). We also investigated the effect of maternal seizures on learning and memory of offspring using the MWM and passive avoidance tests as previous data reported that in addition to adverse effects on maternal and fetal outcome, the occurrence of seizures during gestation might result in cognitive impairment in children born to women with epilepsy (Adab et al., 2004). Exposure to maternal seizure clearly resulted in significant deficits of learning (acquisition) and memory (retention) in the adulthood. At adolescence, learning in the MWM was significantly impaired only on the 5th day, but analysis of retention revealed no significant differences between control and the experimental groups. Adult animals in the control group showed a slightly better spatial performance over the 6 days of training than those exposed to maternal kindling. The prenatal exposure to maternal seizure was also associated with a decrease in distance swum by the adult animals in the target quadrant, suggesting that exposure during gestation to maternal seizure results in subsequent memory impairment. Taken together, the MWM studies showed deficits in both data acquisition (at adolescence) and retention (at adulthood) as a consequence of prenatal exposure to seizures. The observation that prenatal exposure to seizure only leads to retention deficits in the adult cohort of rats is
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consistent with previous studies demonstrating that earlylife stress induced by seizures can progressively influence hippocampal function during adulthood (Holmes and BenAri, 1998). Brunson et al. (2005) have reported that memory disturbances were minimal in early adulthood but severe in middle-age, suggesting that prenatal PTZ-induced seizures, as well as early life chronic stress evoke latearising hippocampus-mediated memory dysfunction during adulthood. Although our findings suggest that prenatal PTZ-induced seizures disrupt spatial cognition, it is possible that the observed deficits in performance could have been a result of general behavioral or sensorimotor impairment, rather than a result of spatial learning and memory deficits. To investigate these possibilities, a visible platform task was performed. We found that prenatal exposure to seizures did not significantly affect the swim length to escape to the visible platform, a finding that is inconsistent with the idea that disruption of escape to the submerged platform is due to general impairments. Furthermore, in our experiments exposure to maternal seizure during gestation in utero was sufficient to impair spatial performance at adolescence and adulthood, a deficit that is perhaps not readily attributed to simple sensorimotor impairment. We, therefore, conclude that the memory deficits in the MWM are not due to generalized behavioral impairments. This is consistent with previous findings from our laboratory (Omrani et al., 2007) indicating that visible platform training in the MWM was not significantly affected by the PTZ-induced kindling in the male rats. To evaluate the effect of prenatal exposure to epileptic seizure on passive avoidance learning, another group of rats were tested in the shuttle-box apparatus. Our data indicated that the passive avoidance learning of either young or adult rats born to the kindled dams was reduced compared to those born to control dams. Our finding of persistent change in learning and memory following exposure to prenatal PTZ-induced kindling is supported by previous studies (Moshe and Albala, 1982; Holmes et al., 1998, 1999; Huang et al., 1999) demonstrating that the immature brain, like the mature brain, is subject to permanent changes in neuronal network excitability following seizures. The impairment in memory also supports other studies showing that PTZ-induced recurrent seizures in adults and developing rodents disrupt learning and memory as assessed by Morris water maze test (Holmes et al., 2002a,b; Huang et al., 2002). However, Zhu-Ge et al. (2008) reported that chronic amygdala kindled seizures achieved by daily application of electric stimulations on amygdala increased memory retention of passive-avoidance test in rats, and acute seizures impair this action. The discrepancy observed between our finding and those of Zhu-Ge et al. (2008) could be due to the differences in the methods of behavioral testing and seizure induction and age of administration of kindling. The pathophysiological mechanism(s) responsible for the impairment in memory is not yet known. The decrease in memory may be due to an imbalance between excitatory and inhibitory neurotransmission induced by seizures
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(Corda et al., 1992; Rocha et al., 1996) and/or due to seizure-induced hypoxia in the placenta which may result in neuronal migration failure in the hippocampus of the embryo (Baka et al., 2004). PTZ-induced seizures suppress GABA-mediated inhibition in the cortex (Corda et al., 1992; Rocha et al., 1996) and decrease GABAB1 receptor expression and, consequently, decrease PKA expression level in primary culture of prenatal rat hippocampal neurons, which may be associated with reduced brain mass and neurobehavioral disturbances during early brain development. These changes may affect learning and memory in the adult life (Naseer et al., 2009). It is also known that the hippocampus plays an important role in the process of learning and long-term memory. It has been reported that the hippocampal cortical laminar structure of a newborn rat from an epileptic mother was consistent with the late embryonic phase and this structure was completely different from that of adults (Baka et al., 2004). Moreover, PTZ-induced seizures resulted in apoptotic neurodegeneration in primary culture of prenatal rat hippocampal neurons (Naseer et al., 2009). Thus, some disorders of learning and memory in adult life may be a consequence of impaired hippocampal migration, maturation and/or neurogeneration during the embryonal period. Manent et al. (2007), however, did not observe any microdysplasia or cortical pathological abnormalities in offspring of kindled rats. Animals born to epileptic dams that kindled intracranially once a day through stimulation of the amygdala from day 15–20 of the gestation were not different from control animals in terms of neurodegeneration, indicating that maternal seizures, at least during the last week of gestation, did not induce cell death in the fetal brain. In addition to the hippocampus, several other brain structures are involved in memory consolidation, including amygdala (McGaugh, 2002), septum (Izquierdo et al., 1992), striatum (Watson and Stanton, 2009) and globus pallidus (Hernadi et al., 1997). Therefore, one can envision that PTZ-induced kindling during pregnancy may affect these fetal brain structures in such a way that it may cause learning and memory impairment in adolescence and adulthood. Further studies are needed to elucidate the underlying mechanisms.
CONCLUSION In summary, our results show that prenatal PTZ-induced kindling produces deficits in learning and memory at adolescence and adulthood using the MWM and passive avoidance tests. It is apparent that subsequent developmental events are not sufficient to overcome the adverse effects of prenatal seizures on brain development. These observations may have clinical implications for cognitive and memory dysfunction associated with epilepsy during pregnancy. However, elucidating the causes of the memory impairment await further studies. Acknowledgments—This work was supported by the Kermanshah University of Medical Sciences.
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(Accepted 2 November 2010) (Available online 9 November 2010)
EFFECT OF PRENATAL PENTYLENETETRAZOL-INDUCED KINDLING ON LEARNING AND MEMORY OF MALE OFFSPRING A. POURMOTABBED,a S. E. NEDAEI,a,b M. CHERAGHI,a S. MORADIAN,a A. TOUHIDI,a M. AEINFAR,a Z. SEYFIa AND T. POURMOTABBEDc*
the other organs (Stumpf and Frost, 1978; Hallak et al., 1999; Lajoie and Mosche, 2004). Most children born to women with epilepsy are normal, but there is an increased risk of abnormal functional neurodevelopment in these children. Two to six percent of children born to an epileptic mother have a developmental delay (Koch et al., 1999) and have 2–7 fold higher rates of mental retardation than controls (Speidel and Meadow, 1972). Recent studies also indicate that generalized tonic– clonic seizures might result in cognitive problems for the child later in life, a phenomenon that is supported by evidence of significantly decreased verbal IQ scores in children whose mothers had more frequent generalized tonic– clonic seizures during pregnancy (Adab et al., 2004). Although, it has been suggested that maternal epilepsy may alter the morphology of the hippocampus of newborns, leading to cognitive impairment (Baka et al., 2004), the effects of epileptic activity on different areas on the brain have not been clearly established (Sederberg-Olsen and Olsen, 1983; Schneiderman et al., 1994). Clinical studies in this area are often difficult to interpret and compare due to exposure of fetus to antiepileptic drugs, possible influence of socio-economical factors, and parental educational level on the outcome of exposed children. Experimental animal studies can be useful in investigating the effects of seizures on neurodevelopment and in identifying the potential mechanisms involved. A periodic systemic injection of a convulsant drug, such as pentylenetetrazol (PTZ) (Assouline et al., 1984) has been shown to induce seizures in animal (Kilbey et al., 1979). PTZ is a noncompetitive antagonist of GABAA receptors. PTZ induced kindling is an accepted animal model for the study of epilepsy and its consequences on memory (Chen et al., 2003). Lamberty and Klitgaard (2000) have reported that PTZ-induced kindling disrupts spatial memory. It has been shown that kindling induced seizures cause hippocampal atrophy and neuronal loss in the limbic area (Cavazos and Sutula, 1990; Pitkanen et al., 1998), leading to learning deficits and cognitive impairment in the animal (Hamm et al., 1995; Gilbert et al., 2000; Mortazavi et al., 2005; Omrani et al., 2007), the effect of maternal seizure on the cognitive performance of newborns is, however, unknown. In this report we examined the effects of prenatal exposure to the maternal tonic-clonic seizure on the learning and memory of adolescents and adult male rats. We found, for the first time, that offspring born to epileptic mothers have significantly impaired spatial memory and passive avoidance learning compared to those born to healthy mother. These findings, thus, provide a mechanistic link
a Department of Physiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Islamic Republic of Iran b Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Islamic Republic of Iran c Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN, USA
Abstract—Current data concerning the effects of maternal seizure during pregnancy on newborns are limited. This study was carried out to investigate the effect of prenatal pentylenetetrazol (PTZ)-induced kindling on learning and memory of offspring. Female Wistar rats were kindled with i.p. injections of 25 mg/kg of PTZ on day 13 of their pregnancy. The spatial performance and passive avoidance learning of pups were tested at 7 weeks and 12 weeks of age using Morris water maze (MWM) task and shuttle-box apparatus, respectively. We found, for the first time, that prenatal exposure to maternal seizure induced by PTZ leads to a significant impairment of learning and memory. In addition, the number of live birth was significantly lower in kindled rats compared to control. In MWM studies, the young offspring of kindled rats had poor spatial learning ability. The frequent tonicclonic seizures in pregnancy was also associated with a poor memory as evidenced by decrease in distance swam in the target quadrant by the offspring of the kindled mother in the adulthood. Data obtained from shuttle-box studies showed that retention latencies of pups born to kindled dams were significantly reduced compared to those born to control dams. The hippocampus, amygdala and frontal cortex are very important for memory consolidation and our data suggest that subsequent developmental events are not sufficient to overcome the adverse effects of prenatal exposure to maternal seizures to these regions of the brain. These observations may have clinical implications for cognitive and memory dysfunction associated with epilepsy during pregnancy. Published by Elsevier Ltd on behalf of IBRO. Key words: prenatal seizure, pentylenetetrazol, Morris water maze, shuttle-box, learning and memory.
Epilepsy is one of the most common chronic diseases affecting over 1 million woman of child bearing age in the USA (Anderson et al., 1999; Bittigau et al., 2003). The infants of epileptic mothers are in higher risk for a variety of adverse pregnancy outcomes. Generalized tonic-clonic seizures increase the risk for hypoxia and acidosis, which can lead to irreversible damage to the CNS as well as to *Corresponding author. Tel: ⫹1-901-4484361; fax: ⫹1-901-4487360. E-mail address: [email protected] (T. Pourmotabbed). Abbreviations: ANOVA, analysis of variance; MWM, Morris water maze; PTZ, pentylenetetrazol. 0306-4522/11 $ - see front matter. Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2010.11.001
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between maternal epilepsy and prenatal brain development and reveal a new aspect of PTZ modulation on learning and memory.
EXPERIMENTAL PROCEDURES In vivo experiments All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of the Laboratory Animals and approved by a local Animal Ethics Committee. All efforts were made to minimize the number of animals used and their suffering. Male and female Wistar rats (Razi Institute, Tehran, Islamic Republic of Iran) aged 3– 4 months and weighting 250 –300 g at the beginning of the experiments were used. The rats were kept in animal facility room (20 –22 °C) on the 12-h light/dark cycle (lights on at 07:00 AM). Animals were housed singly in 24⫻24⫻45 cm3 transparent, plastic cage, with food and water provided ad libitum. Female rats were randomly paired with males one day after delivery to the animal care facility. Vaginal smears were performed on the females every morning (07:00 – 08:00 AM) to look for the presence of sperm. The presence of sperm marked day 1 of pregnancy (Edwards et al., 2002). Each pregnant rat was transported to her home cage and stored singly.
Kindling To test the impact of prenatal seizure on memory, on embryonic day 13 (E13) (E0 being the day in which positive vaginal smear was observed) (Baka et al., 2004), the pregnant dams were divided into two groups consisting of 10 animals each. Animals in the first group were kindled by i.p. injections of 25 mg/kg PTZ (sigma, St. Louis, MO, USA), 1 ml/kg body-weight every 15 min until seizures occurred. The total dosage of PTZ, however, did not exceed 75 mg/kg (three injections) (Klioueva et al., 2001). Pilot study showed that this dose of PTZ injected every 15 min is optimal for kindling. Immediately after injection, seizure activity was observed for 45 min and scored according to Racine (1972), modified by Becker et al. (1992), as follows: stage 0, no response; stage 1, ear and facial twitching; stage 2, myoclonic jerks without rearing; stage 3, myoclonic jerks, rearing; stage 4, turn over into side position, clonic-tonic seizures; stage 5, turn over into back position, generalized clonic-tonic seizures. Animals were considered to be kindled after reaching stage 4 or 5 seizures. The animals in the second group (the control group) received an equivalent volume of normal saline on a similar schedule. After parturition, pups were counted for each litter and weighted. Pups were then housed with their littermates until weaning at postnatal day 21 (Huang et al., 2002). Male pups from each litter were stored singly in a cage. To reduce possible litter effects, a total of four male pups from any litter were used in this experiment. Animals in each group, that is, kindled (K) and the control (C) groups were then divided into two subgroups of 16 males each according to Ehman et al. (2007). The first subgroups were allowed to grow to ⬃7 weeks (K7 and C7; adolescents) and the second subgroups were allowed to grow to 12 weeks of age (K12 and C12; adult). Within each of these subgroups, half of the rats were used for testing in the Morris water maze (MWM), while the other half was tested in the shuttle-box apparatus.
Behavioral assessment Morris water maze (MWM) apparatus. The MWM consisted of a dark circular pool of 140 cm in diameter and 70 cm in height. The pool was filled to a height of 35 cm of water, 22⫾2 °C. A transparent Plexiglas escape platform (10 cm in diameter) was located below the water surface. Pilot experiment showed that platform was not invisible to the rats. The apparatus was located
in a room with numerous extramaze cues that remained constant throughout the experiment. The distance swum to the platform (swim length), and the distance spent in each quadrant was recorded by a video tracking system. Procedure. The MWM procedure was done according to Omrani et al. (2007). Briefly, on the first day, rats were placed on the escape platform for 60 s, which was at the center of the empty pool and on the second day the rats were placed again on the platform in the same position with the pool being full of water. When the rat climbed off the platform, it was guided back on to the platform. Training started the following day. During this period, the escape platform was located in the center of the northwest quadrant and all rats were given a daily session of four trials for six consecutive days. On each trial the rat was placed in the water facing the pool wall at one of four randomly determined starting locations (north, west, east or south poles). Once the rat located the platform, it was allowed to stay on it for 30 s. If the rat was not able to find the platform within 60 s, it was guided to it and allowed to remain on it for 30 s. The rat was then returned to its heated cage for a 30 s inter-trial interval. Twenty-four hours after last training trial, spatial memory was examined in a probe trial. During this trial, the platform was removed from the pool and the rats were allowed to swim freely for 60 s. The distance spent swimming in the quadrant where the platform was previously located was recorded. To assess whether any motivational factors interfered with the rat’s ability to escape, 24 h after probe test, a visible platform trial was designed in which escape could be guided by proximal rather than distal spatial cues (Omrani et al., 2007). During this trial, the platform was raised above the water surface and placed in the southeast quadrant and extra maze cues were removed from the walls and the rats were allowed to swim freely for 60 s. The distance to platform (swim length) was recorded (Gilbert et al., 2000). Passive avoidance apparatus. The passive avoidance apparatus consisted of two light (Plexiglas) and dark (Black) compartments of the same size (20⫻20⫻30 cm3) separated by a door. The floor of the dark compartment was made of stainless-steel bars (0.5 cm diameter) separated by a distance of 1 cm. Intermittent electric shocks (50 Hz, 3 s), 1 mA intensity, were delivered to the floor of the dark compartment by an isolated stimulator (JafariSabet, 2006). Inhibitory-avoidance training. The rats were allowed to become familiar with the laboratory environment 1 h before each of the training or testing sessions. All training and testing were carried out between 08:00 AM and 12:00 AM. Each animal was placed in the light compartment for 20 s, after which the door was raised and the time the animal waited before crossing to the dark (shock) compartment was recorded as the latency. The animal was removed from the experiment when it waited for more than 180 s to cross to the other side. Once the animal completely crossed to the next compartment, the door was closed and a 1 mA foot shock was delivered for 3 s. The rat was then removed from the apparatus and 2 min later, the procedure was repeated. Training was terminated when the rat remained in the light compartment for 120 consecutive seconds. All the animals were trained with a maximum of two trials (Jafari-Sabet, 2006). Retention test. Twenty-four hours after training, a retention test was performed to examine long-term memory. Each animal was placed in the light compartment for 20 s, the door was opened, and the latency for entering into the shock compartment (as described in the training session) was measured as step through latency (STL). During these sessions, no electric shock was applied and the test session ended when the animal entered the shock compartment or remained in the light compartment for 600 s (criterion for retention) (Jafari-Sabet, 2006).
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Statistical analysis All data are presented as mean⫾SEM. Significance of the distance to platform (swim length) in MWM were assessed by twoway analysis of variance (ANOVA) with repeated measures and followed by Tukey’s test for multiple comparisons. Values for the probe trial were compared by one-way ANOVA and followed by Tukey’s test for multiple comparisons. Significance of the mean of the STL in the shuttle box was determined by the Student t-test. Differences were considered significant at the level of P⬍0.05 for all tests.
RESULTS Control animals that received saline instead of PTZ never experienced seizure activity. The female rats that were subjected to PTZ developed the characteristic features of seizure, whose severity reached stage 4/5 of kindling. PTZ-induced kindling significantly affected the number of live birth in kindled animals. Kindled rats gave birth to 9⫾0.53 (mean⫾SD) pups compared to 11⫾0.92 (mean⫾SD) pups born to control animals. This difference was statistically significant (P⬍0.001). However, there was no significant difference in pup’s body weight. The average weight of pups born to control and kindled rats were 6.6⫾0.1 g and 6.4⫾0.2 g, respectively. Learning performance in the MWM
Fig. 1. The effect of prenatal PTZ-induced kindling on spatial performance of male rats at adolescence and adulthood. Mean⫾SEM distance traveled to the platform in the water maze for each of the consecutive 6 d of training for 7-week-old (c7, n⫽8; k7, n⫽8) (A) and 12-week-old (c12, n⫽8; k12, n⫽8) (B) animals. At adolescence, significant impairment occurred only on the 5th day (* P⬍0.05). (C, D) Probe trial at adolescence and in adulthood. Each graph shows the mean percentage (⫾ SEM) of total distance swum in each quadrant for controls (c7 and c12) and kindled (k7 and k12) groups.# P⬍0.05 and
The MWM was used to assess the spatial performance and, as shown in Fig. 1, both 7-week and 12-week old rats learned to find the hidden platform, and escape onto it. Distances swum to the platform decreased over the six testing days in all experimental groups (c7, F5,35⫽9.27, P⬍0.001; c12, F5,35⫽11.74, P⬍0.001; k7, F5,35⫽3.78, P⬍0.01; and k12, F5,35⫽2.4, P⬍0.05). A two-way repeated ANOVA applied to swim lengths of 7-week-old rats found a significant group (F1,14⫽9.49, P⬍0.01) and days (F5,70⫽10.09, P⬍0.001) effects, but no groups and days interaction (F5,70⫽0.22, P⫽0.95). Post hoc comparison showed significant difference only on the 5th day (P⬍0.05) (Fig. 1A). When we tested the spatial performance of 12-week-old animals, we found a trend toward increased swim length in the k12 group of rats (group effect, F1,14⫽3.28, P⫽0.09; days effect, F5,70⫽8.09, P⬍0.001; interaction of group and days, F5,70⫽0.18, P⫽0.96) (Fig. 1B). In our experiments, spatial memory formation was measured by the percentage of distance swum in the target quadrant during probe trial. The probe test result for the k7 group is compared with that of c7 group in Fig. 1C. The distance swum by the both groups in the target quadrant differed from that of the other quadrants (c7, F3,28⫽4.4, P⬍0.05; k7, F3,28⫽5.9, P⬍0.01). The rats that were exposed to maternal seizure also demonstrated a bias for the target quadrant during the probe trial similar to that of the control group (P⬎0.05). When the performance of 12-week-old animals were tested, we found that rats in
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P⬍0.0001 for each quadrant vs. target quadrant. $ P⬍0.05 for target quadrant of k12 group vs. that of c12. The dashed line refers to chance level (25%).
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Fig. 2. The effect of prenatal PTZ-induced kindling on the passive avoidance learning. Mean⫾SEM step through latency significantly reduced in kindled groups compared to the controls either in 7-weekold (c7, n⫽8; k7, n⫽8) (A) or 12-week-old (c12, n⫽8; k12, n⫽8) (B) animals. * P⬍0.05 for kindled groups vs. controls.
the both groups swam statistically more distance in the target quadrant than in the other quadrants (c12, F3,28⫽11.97, P⬍0.0001; K12, F3,28⫽5.13, P⬍0.01). On the other hand, the distance swum in the target quadrant by the animals exposed to maternal seizure (kindled group) was significantly shorter compared with control group (P⬍0.05) (Fig. 1D). However, comparing performance of different groups on a visible platform trial showed no significant differences in swim lengths (P⬎0.05) (data not shown). Passive avoidance The step-through passive avoidance test was also used to evaluate learning and memory. Fig. 2 shows the effects of exposure to prenatal PTZ-induced kindling on memory retention of passive avoidance learning at adolescence (A) and adulthood (B). The data indicate that retention latencies of pups born to the kindled dams were significantly reduced compared to those born to control dams (P⬍0.05).
DISCUSSION Most epileptic woman can become pregnant; however, their pregnancies are prone to more complications with a
high risk of adverse effects on the fetal brain and development (Lajoie and Mosche, 2004). To date, very few well clinical trials or animal studies have addressed these issues. The present study clearly demonstrates that the exposure of the rat fetus to maternal PTZ-induced seizure has deleterious consequences on the learning and memory as indicated by water-maze and passive-avoidance tests. In the current study, pregnant animals received maximum three repetitive PTZ injections on day 13 of gestation. This stage of pregnancy was chosen for induction of kindling to specifically assess the effect of any potential maternal seizure-induced alteration of the brain construction associated with defects in neurogenesis or neuronal migration on memory of the offspring. According to Hatten (1993, 1999) neuronal proliferation in pups is continuing at this time and the epileptic seizures experienced on the 13th day of pregnancy result in migration failure of hippocampal neurons (Baka et al., 2004). In the present study, we have found that the number of live birth was significantly reduced in the kindled compared to control dams. This is consistent with the idea presented by Morrell and Montouris (2004), that seizures may lead to stimulation or inhibition of the hypothalamus, depending on the region affected by seizure and hence on endocrine disorders such as hypothalamic-pituitary-gonadal axis, leading to a reduced number of live births. There is some evidence that epileptic women give birth to children with reduced birth weight (for review see Hvas et al., 2000). In our study, however, there was no difference in body weight of the pups born to the kindled dams compared to those born to the control dams. Thus, gross intrauterine development did not seem to be influenced by whether the dam was epileptic or not as previously reported (Raffo et al., 2009). We also investigated the effect of maternal seizures on learning and memory of offspring using the MWM and passive avoidance tests as previous data reported that in addition to adverse effects on maternal and fetal outcome, the occurrence of seizures during gestation might result in cognitive impairment in children born to women with epilepsy (Adab et al., 2004). Exposure to maternal seizure clearly resulted in significant deficits of learning (acquisition) and memory (retention) in the adulthood. At adolescence, learning in the MWM was significantly impaired only on the 5th day, but analysis of retention revealed no significant differences between control and the experimental groups. Adult animals in the control group showed a slightly better spatial performance over the 6 days of training than those exposed to maternal kindling. The prenatal exposure to maternal seizure was also associated with a decrease in distance swum by the adult animals in the target quadrant, suggesting that exposure during gestation to maternal seizure results in subsequent memory impairment. Taken together, the MWM studies showed deficits in both data acquisition (at adolescence) and retention (at adulthood) as a consequence of prenatal exposure to seizures. The observation that prenatal exposure to seizure only leads to retention deficits in the adult cohort of rats is
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consistent with previous studies demonstrating that earlylife stress induced by seizures can progressively influence hippocampal function during adulthood (Holmes and BenAri, 1998). Brunson et al. (2005) have reported that memory disturbances were minimal in early adulthood but severe in middle-age, suggesting that prenatal PTZ-induced seizures, as well as early life chronic stress evoke latearising hippocampus-mediated memory dysfunction during adulthood. Although our findings suggest that prenatal PTZ-induced seizures disrupt spatial cognition, it is possible that the observed deficits in performance could have been a result of general behavioral or sensorimotor impairment, rather than a result of spatial learning and memory deficits. To investigate these possibilities, a visible platform task was performed. We found that prenatal exposure to seizures did not significantly affect the swim length to escape to the visible platform, a finding that is inconsistent with the idea that disruption of escape to the submerged platform is due to general impairments. Furthermore, in our experiments exposure to maternal seizure during gestation in utero was sufficient to impair spatial performance at adolescence and adulthood, a deficit that is perhaps not readily attributed to simple sensorimotor impairment. We, therefore, conclude that the memory deficits in the MWM are not due to generalized behavioral impairments. This is consistent with previous findings from our laboratory (Omrani et al., 2007) indicating that visible platform training in the MWM was not significantly affected by the PTZ-induced kindling in the male rats. To evaluate the effect of prenatal exposure to epileptic seizure on passive avoidance learning, another group of rats were tested in the shuttle-box apparatus. Our data indicated that the passive avoidance learning of either young or adult rats born to the kindled dams was reduced compared to those born to control dams. Our finding of persistent change in learning and memory following exposure to prenatal PTZ-induced kindling is supported by previous studies (Moshe and Albala, 1982; Holmes et al., 1998, 1999; Huang et al., 1999) demonstrating that the immature brain, like the mature brain, is subject to permanent changes in neuronal network excitability following seizures. The impairment in memory also supports other studies showing that PTZ-induced recurrent seizures in adults and developing rodents disrupt learning and memory as assessed by Morris water maze test (Holmes et al., 2002a,b; Huang et al., 2002). However, Zhu-Ge et al. (2008) reported that chronic amygdala kindled seizures achieved by daily application of electric stimulations on amygdala increased memory retention of passive-avoidance test in rats, and acute seizures impair this action. The discrepancy observed between our finding and those of Zhu-Ge et al. (2008) could be due to the differences in the methods of behavioral testing and seizure induction and age of administration of kindling. The pathophysiological mechanism(s) responsible for the impairment in memory is not yet known. The decrease in memory may be due to an imbalance between excitatory and inhibitory neurotransmission induced by seizures
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(Corda et al., 1992; Rocha et al., 1996) and/or due to seizure-induced hypoxia in the placenta which may result in neuronal migration failure in the hippocampus of the embryo (Baka et al., 2004). PTZ-induced seizures suppress GABA-mediated inhibition in the cortex (Corda et al., 1992; Rocha et al., 1996) and decrease GABAB1 receptor expression and, consequently, decrease PKA expression level in primary culture of prenatal rat hippocampal neurons, which may be associated with reduced brain mass and neurobehavioral disturbances during early brain development. These changes may affect learning and memory in the adult life (Naseer et al., 2009). It is also known that the hippocampus plays an important role in the process of learning and long-term memory. It has been reported that the hippocampal cortical laminar structure of a newborn rat from an epileptic mother was consistent with the late embryonic phase and this structure was completely different from that of adults (Baka et al., 2004). Moreover, PTZ-induced seizures resulted in apoptotic neurodegeneration in primary culture of prenatal rat hippocampal neurons (Naseer et al., 2009). Thus, some disorders of learning and memory in adult life may be a consequence of impaired hippocampal migration, maturation and/or neurogeneration during the embryonal period. Manent et al. (2007), however, did not observe any microdysplasia or cortical pathological abnormalities in offspring of kindled rats. Animals born to epileptic dams that kindled intracranially once a day through stimulation of the amygdala from day 15–20 of the gestation were not different from control animals in terms of neurodegeneration, indicating that maternal seizures, at least during the last week of gestation, did not induce cell death in the fetal brain. In addition to the hippocampus, several other brain structures are involved in memory consolidation, including amygdala (McGaugh, 2002), septum (Izquierdo et al., 1992), striatum (Watson and Stanton, 2009) and globus pallidus (Hernadi et al., 1997). Therefore, one can envision that PTZ-induced kindling during pregnancy may affect these fetal brain structures in such a way that it may cause learning and memory impairment in adolescence and adulthood. Further studies are needed to elucidate the underlying mechanisms.
CONCLUSION In summary, our results show that prenatal PTZ-induced kindling produces deficits in learning and memory at adolescence and adulthood using the MWM and passive avoidance tests. It is apparent that subsequent developmental events are not sufficient to overcome the adverse effects of prenatal seizures on brain development. These observations may have clinical implications for cognitive and memory dysfunction associated with epilepsy during pregnancy. However, elucidating the causes of the memory impairment await further studies. Acknowledgments—This work was supported by the Kermanshah University of Medical Sciences.
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(Accepted 2 November 2010) (Available online 9 November 2010)