• Pentylenetetrazol;
  • Seizures;
  • Stress;
  • Long-term consequences;
  • Learning and memory;
  • cAMP-responsive element-binding protein


  1. Top of page
  2. Abstract

Summary:  Purpose: Maternal deprivation is stressful for the neonate. The aim of this study was to investigate the short- and long-term effects of maternal separation on recurrent seizures in the developing brain.

Methods: Rats were divided into four groups according to whether the rat pups were treated with maternal deprivation from postnatal day 2 (P2) to P9 or neonatal seizures induced by intraperitoneal (i.p.) injection of pentylenetetrazol (PTZ) from P10 to P14. Rats in the control group received saline i.p. injection from P10 to P14; rats in the isolation group underwent daily separation from their dams from P2 to P9; rats in the PTZ-treated group were subjected to PTZ-induced recurrent seizures from P10 to P14; rats in the isolation plus PTZ–treated group were subjected to maternal deprivation from P2 to P7 followed by serial seizures from P10 to P14. In addition, subsets of rats at P15 were killed and the brains assessed for acute neuronal degeneration. Visual–spatial memory test using the Morris water maze task was performed at P80. After testing, the hippocampus was evaluated for histologic lesions and cyclic adenosine monophosphate (cAMP)-responsive element-binding protein phosphorylation at serine-133 (pCREBSer-133), an important transcription factor underlying learning and memory.

Results: All rats given PTZ developed recurrent seizures. After PTZ administration, rats with a history of maternal deprivation had more intense impairment than did rats with maternal deprivation and neonatal seizures than those without deprivation. Neuronal degeneration was most prominent in the rats exposed to maternal deprivation plus recurrent seizures. Rats receiving maternal deprivation or PTZ-induced recurrent seizures exhibited only spatial deficits, but no morphologic changes in the hippocampus. However, rats with maternal deprivation plus PTZ-induced recurrent seizures exhibited worse visual–spatial learning compared with rats with either isolation or PTZ-induced recurrent seizures alone. The levels of pCREBSer-133 may play a role in the decrease in the hippocampus from the rats subjected to maternal deprivation and/or PTZ-induced recurrent seizures, as compared with rats exposed to vehicle-control saline. These results indicate that repeated maternal deprivation can exacerbate long-term cognitive deficits resulting from neonatal seizures. In addition, impaired phosphorylation of CREBSer-133.

Conclusions: Repeated maternal deprivation stress has synergistic effects with recurrent seizures in inducing neurologic damage in the developing brain.

Seizures occur more frequently in the neonatal period and early childhood than at any other time in life. In rodent studies, recurrent neonatal seizures result in long-term cognitive deficits (1–3), reduced dentate granule cell neurogenesis (4), and synaptic reorganization in the terminal field of the mossy fiber pathway (1–3).

Neonatal physiology and development are regulated to a great extent by mother–child interactions. In animal studies, environmental manipulation during the early postnatal period induces a decrease in anxiety-like behavior in adulthood (5). Maternal deprivation (neonatal isolation) appears to be stressful to the pup. Isolation of the rat pups from their mother, for even a brief period, evokes vocalizations (6) and activates the hypothalamic– pituitary–adrenal (HPA) axis (7), demonstrating that the separation experience is stressful for the neonate. If the isolation experience is repeated, enduring effects including behavioral abnormalities (7,8), neurochemical and endocrinologic changes (8,9), alterations of hippocampal neuroplasticity (10), and infralimbic cortex synaptic connections (11) can be observed in adulthood.

Little information is available in the literature regarding the molecular and cellular mechanisms that might contribute to memory deficits after seizures in the developing brain. Understanding the cellular mechanisms underlying memory deficits after seizures may open new therapeutic avenues for these transcriptional factor, is critically required in synaptic plasticity, learning and memory (12–14). CREB is a member of a large family [CREB/ATF (activating transcription factor)] of structurally related proteins that bind to the CRE promoter. CREB can be activated seconds or minutes after external stimulation through an intracellular increase in cyclic adenosine monophosphate (cAMP) or calcium. cAMP then activates the catalytic subunit of protein kinase A (PKA), by releasing its binding to the regulatory subunit. The catalytic subunit passively translocates to the nucleus where it phosphorylates one or more CREB-related transcription factors that activate the transcription of genes that lead to synaptic plasticity (15). Ser-133 phosphorylation is considered to be a critical event that mediates the initiation of transcription.), an important patients. A growing body of evidence suggests that phosphorylation of cAMP response element-binding protein at serine-133 (pCREBSer-133).

Support for the role of CREB in memory comes from models varying from the fruit fly to rodent (15–17). CREB activation occurs in the formation of new memories in Drosophila(18–20), Aplysia(21–23), and mice (12,18,24). Long-term memory and long-term potentiation is defective in CREB mutant mice (25). Spaced electrical tetani, which are sufficient to induce long-term potentiation in hippocampal slices from mice, also induce CREB-mediated gene expression (26). Antisense oligonucleotides specific for CREB transcripts injected into the hippocampus have been shown to impair water maze performance (27). Similarly, Lamprecht et al. (28) disrupted long-term memory of conditioned taste aversion by injecting antisense oligonucleotides into the amygdala. Thus it is possible that disrupted pCREBSer-133 function may be instrumental in the memory deficit after seizures during certain environmental challenges, such as maternal deprivation, during the neonatal period.

In most published studies, seizures have been induced in the developing animals under normal housing conditions. However, in clinical situations, neonates with seizures may also be in a stressful environment, such as the neonatal intensive care unit where the infants are separated from the mother for prolonged periods. In this study, we (a) examined whether maternal deprivation aggravated long-term cognitive effects after pentylenetetrazol (PTZ)-induced recurrent seizures, a conventional model for inducing seizures (2,3), in immature animals; and (b) explored the possible mechanisms underlying the memory deficits after recurrent seizures in the presence or absence of isolation stress in the developing brains. We found that maternal deprivation accentuated not only the immediate neuronal injury but also long-term seizure-induced cognitive deficits.


  1. Top of page
  2. Abstract


Sprague–Dawley (SD) male rats were used throughout the experiments. Pregnant rats were purchased from the National Science Counsel and housed in Chang Gung Memorial Hospital, Kaohsiung, Taiwan. The birth data were designated postnatal day 0 (P0). Litters were reduced to 12 at P1. Rat pups were weaned at P21 and grouped in plastic cages on a standard 12/12-h light/dark cycle. The present study was in accordance with the experimental guideline of National Science Counsel, Taiwan, and all efforts were made to minimize the number of rats.

Maternal deprivation (neonatal isolation) procedure

Rat pups were placed in individual round plastic cups (9.5 cm diameter × 8 cm deep) containing no bedding and were isolated 1 h/day under a lamp to maintain body warmth. At the end of the isolation period, pups were returned to the nest and the dam. Isolations occurred between P2 and P9. Isolations were carried out between 9 a.m. and 10 a.m. each day, and rat pups were weighed every day.

Induction of seizures

Rats were divided into four groups based on whether rats were subjected to maternal deprivation or PTZ-induced seizures. We list the groups in numerical order:

  • 1
    Control group (n = 8): pups received intraperitoneal (i.p.) saline from P10 to P14 without maternal deprivation;
  • 2
    Isolation group (n = 9): pups exposed to an individual period of isolation from P2 to P9 and were then administered saline i.p. from P10 to P14 (isolation only);
  • 3
    PTZ-treated group (n = 12): pups remained undisturbed with their families and were then administered PTZ i.p. from P10 to P14 (seizures only);
  • 4
    Isolation plus PTZ–treated group (n = 12): pups exposed to isolation from P2 to P9 and were then administered with PTZ i.p. from P10 to P14.

PTZ (Sigma, St. Louis, MO, U.S.A.), a γ-aminobutyric acid subtype A (GABAA)-receptor antagonist, was used to induce seizures, as previously described (3). A dose of 90 mg/kg was injected i.p., followed 10 min later by an injection of 40 mg/kg. Thereafter, every 10 min, additional doses of 20 mg/kg were administered to the rat until the onset of status epilepticus (SE), which was characterized by clonic movements of the four limbs and loss of posture. Seizure duration and mortality rate were recorded regularly throughout the entire study.

Silver impregnation stain and quantitation

Degeneration of neurons was assessed 24 h after the fifth PTZ seizures (P15) by using silver-impregnation techniques (29–31). Four to five rat pups from each group were perfused with 0.9% saline solution, followed by 4% PFA. The brains were cut coronally through the entire hippocampus at 30 μm on a freezing microtome. Brain tissues were washed 3 times for 5 min with distilled H2O, followed by pretreatment (equal volume of 9% NaOH and 1.2% NH4NO3) 2 times for 5 min and impregnation solution (150 ml 9% NaOH; 100 ml of 16% NH4NO3; and 1.5 ml of 50% AgNO3) for 10 min. The tissue was then rapidly washed in washing solution (1 ml of 1.2% NH4NO3 added to 100 ml of solutions containing 5 g of anhydrous Na2CO3; 300 ml of 95% ethanol; brought to 1:l with distilled water) 3 times within 5 min, followed by immersion in developing solution (1 ml 1.2% NH4NO3 with 100 ml of 0.05 g anhydrous citrate acid in 15 ml of 37% formalin, 100 ml 95% ethanol, and 700 ml H2O adjusted to pH 5.8–6.2 with 9% NaOH, and finally brought to 1:l with H2O) for ≥1 min. Sections were then mounted onto gelatin-dipped slides. After drying, sections were placed in three changes of 0.5% acetic acid for 10 min each, washed with H2O, dehydrated in a series of ethanol (70–100%), cleared with butanol and three changes of xylene, and coverslipped.

A semiquantitative, 4-point scale was modified from Bartus et al. (30) and was implemented to analyze the changes of silver staining in the hippocampus. Four hippocampal sections per rat were independently examined and rated by two trained technicians, and a mean score for each rat was computed from the two raters' scores. The scales used for visual analysis were addressed as follows: 0, normal cells with no silver staining of pyramidal cells or dentate granular cells; 1, slight damage as indicated by patchy light gray to dark black staining of pyramidal cells and dentate granular cells; 2, extensive and more intense argyrophilic somata of pyramidal cells and dentate granular cells; 3, severe cell damage as evidenced by nearly complete argyrophilic somata and both the apical and basilar dendritic fields throughout the entire CA1 and CA3 regions and dentate gyrus.

Morris water maze task

Animals were evaluated for spatial memory in the Morris water maze between P80 and P85. Morris water maze task was performed as previously described (3). A pool (180 cm diameter × 50 cm high) was filled with water (26 ± 1°C) to a depth of 25 cm, and a 10 × 10-cm plexiglas platform was positioned in the center of one fixed quadrant 1 cm below the water surface. The water was made opaque by addition of 100 ml evaporated milk to prevent visualization of the platform. A video camera was set above the center of the pool and connected to a videotraction system (EthoVision; Noldus, The Netherlands). Several visual cues were placed around the testing room to enable the rats to learn the platform location. On the first day, each rat was placed in the pool for 60 s without the platform present; this free swim enabled the rat to become habituated to the training environment. On days 2 to 5, each rat was trained for 24 trials (six trials per day) to locate and escape onto the submerged platform. On mounting the platform, the rats were given a 30-s rest period, after which the next trial was started. If the rat did not find the platform in 120 s, it was manually placed on the platform. Latencies to escape onto the platform, distance traveled, and average swimming speed were recorded. On day 6, the platform was removed. The rat was allowed 60 s of free swimming. The time spent in the quadrant where the platform was previous located was measured (probe trial). The testing procedure for locating the hidden platform is considered a measure of spatial reference memory, whereas the probe trial was considered to measure the strength of spatial learning (32).

Hippocampal slice preparation

After the Morris water maze task, animals were decapitated, and the hippocampal tissues were quickly removed. Hippocampal slices were prepared as described previously (33). Hippocampal slices (400 μm) were transversely cut with a vibroslicer (Campden Instruments, Sileby, Loughborough, UK) and were immediately transferred to the artificial cerebrospinal fluid (aCSF) in an incubating chamber provided with humidified 95% O2/5% CO2 gas at room temperature. After an incubation period of ≥1 h, the slice was transferred to a submerged-type constant flow-recording chamber (approximate volume, 1.0 ml) perfused at a rate of ∼1.5–2.0 ml/min with oxygenated aCSF (95% O2/5% CO2) at 30.0 ± 0.5°C.

The control aCSF consisted of (in mM): NaCl (124), KCl (3.5), CaCl2(2), MgCl2(1), NaH2PO4 (1.25), NaHCO3(26), d-glucose (10), at pH 7.4. The osmolarity of the solutions was kept at 305 ± 5 mOsm. In the present study, all hippocampal slices were treated with glutamate (500 μM), which served as a conventional synaptic stimulus for the activation of molecular signals leading to phosphorylation of CREBSer-133(14).

Preparation of protein extracts and Western blots

Hippocampal tissue slices were immediately frozen at –20°C and were homogenized to obtain nuclear protein extracts and cytoplasmic supernatant as previously described (34). The protein concentrations were measured by the use of the Bio-Rad DC protein assay kits (Cat. no. 500-0112; Bio-Rad Laboratories, Hercules, CA, U.S.A.). A volume of 2× sample buffer was added to the homogenate, and the sample was incubated in 95°C water bath for 10 min. Samples were loaded on the 10% sodium dodecylsulfate (SDS)–polyacrylamide gels and resolved by standard electrophoresis (Novex, Carlsbad, CA, U.S.A.). The gels were then transferred onto PVDF filters. The filters were incubated with polyclonal anti-phospho-CREBSer-133 (1:1,000; Upstate Biotechnology, Lake Placid, NY, U.S.A.) and were visualized by chemiluminescence. Then the filters were stripped and reprobed with anti-CREB (1:1,000, Upstate Biotechnology) to detect total CREB. For quantitation of immunoblot signals, the band intensity was measured with Kodak Digital Science 1D program (Rochester, NY, U.S.A.). Each phospho-CREBSer-133 band intensity was equalized to the total CREB (1:1,000, Upstate Biotechnology) signal in the same lane. The increases in phosphorylation of CREBSer-133 were normalized to the vehicle-control level and expressed as fold increase.


At the completion of the experiments, the animals were killed, and coronal sections through the entire hippocampus were cut at 30 μm on a freezing microtome. Every fourth section was stained with cresyl violet and analyzed for cell loss.

Statistical methods

The Morris water maze performance was analyzed by one-way analysis of variance (ANOVA) with post hoc Bonferroni test and repeated measures. The immunoblot intensity and scores for characterizing sliver staining were analyzed by one-way ANOVA with post hoc Bonferroni test. The Kruskal–Wallis test was used to compare the seizure duration. Values were expressed as mean ± SEM, and significance was defined as p < 0.05 for all tests.


  1. Top of page
  2. Abstract


To determine whether maternal isolation caused significant changes in body weight, measurements of body weight were performed at P10. Before PTZ-induced seizures, the body weights of all rat pups isolated for 8 days did not differ from those of their controls (p > 0.1).

Behavioral effects

Neither seizures nor death was elicited in the saline-treated rats. All rats subjected to PTZ developed head bobbing followed by intermittent forelimb clonus and hyperextension of the tails and hindlimbs. The cumulative dose of PTZ needed to induce seizures at P14 was 144.29 ± 3.69 mg in the PTZ-treated group and 146.67 ± 20.28 mg in the isolation plus PTZ–treated group. The seizure duration was compared at P14 and was significantly prolonged in the isolation plus PTZ–treated group (334 ± 26 min) as compared with the PTZ-treated group (220 ± 20 min; p < 0.05).

Silver staining

As shown in Fig. 1, no silver staining was detected in the control group. Scattered argyrophilic stained neurons, demonstrating acute neuronal degeneration, were seen in the dentate gyrus and CA3 subfield from both the isolated and the PTZ-treated rats. Substantially more intense silver staining in CA1, CA3, dentate hilar area, and the dentate gyrus was visible in rats subjected to isolation plus PTZ treatment (Fig. 1H, 1h', 1h”). The results of the silver staining score are as follows: Control, 0.25 ± 0.17; Isolation group, 0.33 ± 0.21; PTZ-treated group, 0.75 ± 0.21; Isolation plus seizure group, 1.5 ± 0.22 (F3, 20 = 7.71, p < 0.05, n = 6 for each group). Post hoc testing showed that there are significant differences between the isolation plus PTZ–treated and the isolation and the control groups.


Figure 1. Representative photomicrographs of hippocampal coronal sections of cresyl violet stained (A–D) and silver stain (E–H) from vehicle controls (A,E,e',e”), isolation (B,F,f',f”), pentylenetetrazol (PTZ)-treated (C,G,g',g”), and isolation plus PTZ-treated (D,H,h',h”) group. Examples of silver staining at high magnification are presented in the dentate gyrus (e'–h') and CA1 subfield (e”–h”). In cresyl violet staining, all the rats showed no neuronal cell loss (A–D). In the isolation plus PTZ–treated group, prominent dense silver-depositing granules in shrunken cytoplasm, indicative of degenerative neurons (arrowheads), are observed in CA1, CA3, dentate hilar area, and the dentate gyrus (H,h',h”), whereas only a few dark silver-stained neurons are visible in both isolation (F,f',f”) and PTZ-treated (G,g',g”) groups. Scale bar for A–H, 300 μm; e'–h', 150 μm; and e”–h”, 75 μm.

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Morris water maze task

Spatial learning was tested by Morris water maze task at P80, and Fig. 2 shows the escape latency in the water maze as a function of day. Although all groups displayed improvements over the 4 days of training, the performance of this task among these groups was significantly different in the first 3 testing days, with the maximum in the first testing day (F3, 37 = 10.01, p < 0.001, Fig. 2). Post hoc testing demonstrated that compared with the controls, both the isolation and the PTZ-treated groups had differences only in the first testing day (p < 0.05), whereas the isolation plus PTZ–treated group showed significant differences in the first 3 testing days (p < 0.05). Rats subjected to isolation plus PTZ treatment exhibited considerable impairments in the task during the second and the third testing days, as compared with the isolation and the PTZ-treated group (p < 0.05). This finding demonstrates that isolation enhances the impairment of spatial learning in the rats subjected to neonatal seizures. There was no significant difference in swimming speed among the control, isolation, PTZ, and isolation plus PTZ–treated groups (p > 0.1). In addition, in the probe trial, there was an increasing percentage of time in the target quadrant in all groups, but the differences did not reach statistical significance (p > 0.1).


Figure 2. Latencies (seconds) to escape in the Morris water maze (mean ± SEM) in the vehicle control (CTL), isolation (ISO), pentylenetetrazol (PTZ)-treated, and isolation plus PTZ–induced seizures (ISO+PTZ) groups. There are significant differences among groups in the first 3 testing days, with the maximum in the first testing day (F3, 37 = 10.01; p < 0.001). Furthermore, post hoc test revealed that compared with the control group, both the isolation and the PTZ-treated groups revealed marked differences only in the first testing day, and rats subjected with isolation plus PTZ treatment exhibited significant impairment in the first 3 testing days (p < 0.05). Furthermore, the rats subjected to isolation plus PTZ treatment exhibited considerable impairments in the task in the second and third testing days, as compared with either the isolation or the PTZ-treated group (p < 0.05). §Statistical difference (p < 0.05) among four groups; *statistical difference (p < 0.05) compared with the control group.

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Decreased CREBSer-133 phosphorylation

To determine whether phosphorylated CREBSer-133 was involved in long-term cognitive deficits after PTZ-induced recurrent seizures in the presence of maternal deprivation during the neonatal period, Western blotting was performed on hippocampal slices. As shown in Fig. 3, pCREBSer-133 levels were decreased in the rats subjected to PTZ-induced recurrent seizures (38.9 ± 6.7%; n = 5; p < 0.05), isolation alone (43.6 ± 7.2%; n = 5; p < 0.05), and isolation plus PTZ treatment (47.6 ± 7.6%; n = 5; p < 0.05), as compared with the rats subjected with vehicle-control saline. There was no significant difference among the groups for the hippocampal expression of pCREBSer133.


Figure 3. A: Representative immunoblots of phosphorylated CREBSer-133 (pCREBSer-133) and total CREB (tCREB). B: Summary of normalized pCREBSer-133 immunoreactivity. *p < 0.05 as compared with the rats subjected with vehicle saline. Isolation plus PTZ compared with either PTZ or isolation alone.

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No histologic lesions were seen, using cresyl violet, in any of the groups (Fig. 1A–D).


  1. Top of page
  2. Abstract

The present study shows that PTZ-induced recurrent seizures in the developing brain lead to long-term cognitive deficit and support the growing evidence that recurrent seizures in the developing brain are harmful (35,36). We demonstrated that both the abilities of spatial memory and pCREBSer-133 were reduced in rats subjected to PTZ-induced recurrent seizures during the neonatal period. Furthermore, this study demonstrates for the first time that repeated neonatal isolation stress exhibits synergism with neonatal seizures in inducing both short- and long-term detrimental effects.

In rodent studies, both prenatal stress (37) and postnatal stress (38) result in decreased neurogenesis in the granule cells of the dentate gyrus. The dentate gyrus has an important role in learning (39) and undergoes the majority of its development during the first 2 weeks of life (40). Repeated neonatal isolation stress caused abnormal development of the functional pathway, in particular in the limbic system, and these changes may persist into adulthood (41). Kehoe and Bronzino (42) demonstrated that repeated neonatal isolation stress produced an alteration of long-term potentiation (LTP) in the adult animals. As shown in this study, intense silver staining of granule cells of the dentate gyrus was detected immediately in rats subjected to maternal isolation plus recurrent seizures. Our results suggest that the dentate gyrus is particularly vulnerable to early environmental manipulations and contributes to long-term cognitive deficits after seizures and isolation. Conversely, cresyl violet staining of adult rat hippocampus showed no neuronal loss in any of the groups. These results suggest that silver staining is a marker of acute neuronal degeneration (30), but not a definite marker, per se, for permanent cell loss (31).

Activation and expression of CRH is developmentally regulated and is associated with hippocampal excitability and seizure generation in the developing brain (43). Repeated isolation results in potentiation of the hypothalamus–pituitary–adrenal (HPA) axis in adulthood (44). Maternal deprivation enhances the HPA response after exposure to a novel environment (45). Furthermore, after repeated neonatal isolation, neurosteroids are altered, as indicated by a reduction of dihydroprogesterone and an increase of allopregnanolone (46). Exposure to high levels of glucocorticoids during this critical period in development has been shown to lead to numerous detrimental effects on the developing central nervous system (47). Prolonged elevations of glucocorticoid levels are toxic to hippocampal neurons by increasing their vulnerability to a variety of insults (48–50). Furthermore, glucocorticoids exacerbate neuronal damage caused by pathophysiologic challenges, including hypoxia–ischemia, traumatic brain injury, and repeated seizures (51,52). We suggest that neonatal stress, through potentiation of the HPA axis and an alteration of neurosteroids, can exacerbate immediate neuronal insults, possibly by alterations of neuron connectivity. Neonatal seizures and stress appear to be synergistic, leading to enduring detrimental effects in cognitive functions.

The possible mechanisms underlying long-term cognitive deficits resulting from various pathophysiologic environments, such as recurrent seizures in the presence of maternal deprivation during the neonatal period, remain unclear. Although there is now a relative agreement of molecular-signaling pathways for certain forms of learning and memory in invertebrates such as Aplysia and Drosophila, the mechanisms that are responsible for learning and memory in mammals remain unclear. Studies of several forms of learning and memory (e.g., behavioral sensitization, Morris water maze task, and classic conditioning in vertebrates) indicate that both behavioral long-term memory and its neural representation require gene expression triggered by pCREBSer-133 that consequently leads to the growth of new synaptic connections (15,18,53,54). Despite the correlation of pCREBSer-133 with performance on the Morris water maze, the failure to show a difference between the isolation, the PTZ-treated, and the combined isolation plus PTZ–treated groups indicates that the absolute deficits in pCREBSer-133 are not the sole factor determining the severity of spatial memory performance. PCREBSer-133-independent mechanisms must be involved in memory impairment after the seizures.

In surgically treated patients with temporal lobe epilepsy, a history of prolonged febrile seizures in childhood can usually be found (55). Conversely, epidemiologic studies showed that the risk of hippocampal sclerosis and temporal lobe epilepsy after an initially provoked or unprovoked seizure was low (56). Mathern et al. (57) and Katzir et al. (58) proposed that secondary physiologic decompensations, such as loss of cerebral autoregulation during seizure attacks, might contribute to the seizure-induced neuronal damage and hence individual variability in long-term outcomes. Here we showed that stress also could exert synergistically detrimental effects on recurrent seizures in the developing brain. Taken together, our results support the concept that, under certain circumstances, the brain is more vulnerable to seizure-induced damage that causes permanent molecular signaling, morphologic, and behavioral changes in the hippocampus.

The demonstration that isolation stress has immediate and enduring effects in association with recurrent seizures in the developing brain may have clinical implications. The environment immediately after birth, which provides important socioemotional experience during the earliest phases of postnatal brain maturation, is the mother–child interaction. Neonates with seizures are usually separated from their mothers, and therapeutic interventions, such as noise, light, endotracheal suctioning, and vein punctures are both painful and stressful. Our findings suggest that psychological and pharmacologic interventions to prevent stress responses in the neonates are needed to reduce aggravating effects of stress on seizure-induced brain damage.

Acknowledgment: The study was supported in part by grants CMRPG8013 from Chang Gung Memorial Hospital, New Century Health Care Promotion Foundation, and NSC 91-2314-B-182A-050 (L-T.H.), 1261 from Chang Gung Memorial Hospital and NHRI-EX91-8909BP (S.N.Y.), a Mental Retardation Research Center grant from NIH (2P30HD18655), and a grant from the NINDS (NS27984) (G.L.H.).


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  2. Abstract
  • 1
    Holmes GL, Gairsa JL, Chevassus-Au-Louis N, et al. Consequences of neonatal seizures in the rat: morphological and behavioral effects. Ann Neurol 1998;44: 84557.
  • 2
    Holmes GL, Sarkisian MR, Ben-Ari Y, et al. Mossy fiber sprouting after recurrent seizures during early development in rats. J Comp Neurol 1999;404: 53753.
  • 3
    Huang LT, Yang SN, Liou CW, et al. Pentylenetetrazole-induced recurrent seizures in rat pups: time course on spatial learning and long-term effects. Epilepsia 2002;43: 56773.
  • 4
    McCabe BK, Silveira DC, Cilio MR, et al. Neonatal seizures result in a decrease in neurogenesis in the dentate. J Neurosci 2001;21: 2094103.
  • 5
    Vallee M, Mayo W, Dellu F, et al. Perinatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone secretion. J Neurosci 1997;17: 262636.
  • 6
    Kehoe P, Blass EM. Opioid-mediation of separation distress in 10-day-old rats: reversal of stress with maternal stimuli. Dev Psychobiol 1986;19: 38598.
  • 7
    Hennessy MB, Moorman L. Factors influencing cortisol and behavioral responses to maternal separation in guinea pigs. Behav Neurosci 1989;103: 37885.
  • 8
    Heidbreder CA, Weiss IC, Domeney AM, et al. Behavioral, neurochemical and endocrinological characterization of the early social isolation syndrome. Neuroscience 2000;100: 74968.
  • 9
    Penke Z, Felszeghy K, Fernette B, et al. Postnatal maternal deprivation produces long-lasting modifications of the stress response, feeding and stress-related behaviour in the rat. Eur J Neurosci 2001;14: 74755.
  • 10
    Kehoe P, Clash K, Skipsey K, et al. Brain dopamine response in isolated 10-day-old rats: assessment using D2 binding and dopamine turnover. Pharmacol Biochem Behav 1996;53: 419.
  • 11
    Ovtscharoff W Jr, Braun K. Maternal separation and social isolation modulate the postnatal development of synaptic composition in the infralimbic cortex of Octodon degus. Neuroscience 2001;104: 3340.
  • 12
    Impey S, Smith DM, Obrietan K, et al. Stimulation of the cAMP response element (CRE)-mediated transcription during contextual learning. Nat Neurosci 1998;1: 595601.
  • 13
    Silva AJ, Kogan JH, Frankland PW, et al. CREB and memory. Annu Rev Neurosci 1998;21: 12748.
  • 14
    Deisseroth K, Heist EK, Tsien RW. Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature 1998;392: 198202.
  • 15
    Bailey CH, Bartsch D, Kandel ER. Toward a molecular definition of long-term memory storage. Proc Natl Acad Sci U S A 1996;93: 1344552.
  • 16
    Lamprecht R. CREB: a message to remember. Cell Mol Life Sci 1999;55: 55463.
  • 17
    Mayr B, Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2001;2: 599609.
  • 18
    Yin JC, Tully T. CREB and the formation of long-term memory. Curr Opin Neurobiol 1996;6: 2648.
  • 19
    Yin JC, Del Vecchio M, Zhou H, et al. CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila. Cell 1995;81: 10715.
  • 20
    Yin JC, Wallach JS, Del Vecchio M, et al. Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 1994;79: 4958.
  • 21
    Kandel ER. The molecular biology of memory storage: a dialogue between genes and synapses. Science 2001;294: 10308.
  • 22
    Dash PK, Moore AN. Characterization and phosphorylation of CREB-like proteins in Aplysia central nervous system. Brain Res Mol Brain Res 1996;39: 4351.
  • 23
    Bartsch D, Ghirardi M, Skehel PA, et al. Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change. Cell 1995;83: 97992.
  • 24
    Williams BM, Luo Y, Ward C, et al. Environmental enrichment: effects on spatial memory and hippocampal CREB immunoreactivity. Physiol Behav 2001;73: 64958.
  • 25
    Bourtchuladze R, Frenguelli B, Blendy J, et al. Deficient long-term memory in mice with a targeted mutation of the cAMP- responsive element-binding protein. Cell 1994;79: 5968.
  • 26
    Impey S, Mark M, Villacres EC, et al. Induction of CRE-mediated gene expression by stimuli that generate long-lasting LTP in area CA1 of the hippocampus. Neuron 1996;16: 97382.
  • 27
    Guzowski JF, McGaugh JL. Antisense oligodeoxynucleotide-mediated disruption of hippocampal cAMP response element binding protein levels impairs consolidation of memory for water maze training. Proc Natl Acad Sci U S A 1997;94: 26938.
  • 28
    Lamprecht R, Hazvi S, Dudai Y. cAMP response element-binding protein in the amygdala is required for long- but not short-term conditioned taste aversion memory. J Neurosci 1997;17: 844350.
  • 29
    Gallyas F, Wilff JR, Bottcher H, et al. A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system. Stain Technol 1980;55: 299306.
  • 30
    Bartus RT, Dean RL, Mennerick S, et al. Temporal ordering of pathogenic events following transient global ischemia. Brain Res 1998;790: 113.
  • 31
    Toth Z, Yan X-X, Haftoglou S, et al. Seizure-induced neuronal injury: vulnerability to febrile seizures in an immature rat model. J Neurosci 1998;18: 428594.
  • 32
    Jeltsch H, Bertrand F, Lazarus C, et al. Cognitive performances and locomotor activity following dentate granule cell damage in rats: role of lesion extent and type of memory tested. Neurobiol Learn Mem 2001;76: 81105.
  • 33
    Yang SN. Sustained enhancement of AMPA receptor- and NMDA receptor-mediated currents induced by dopamine D1/D5 receptor activation in the hippocampus: an essential role of postsynaptic Ca2+. Hippocampus 2000;10: 5763.
  • 34
    Pennypacker KR, Walczak D, Thai L, et al. Kainate-induced changes in opioid peptide genes and AP-1 protein expression in the rat hippocampus. J Neurochem 1993;60: 20411.
  • 35
    Wasterlain CG. Recurrent seizures in the developing brain are harmful. Epilepsia 1997;38: 72834.
  • 36
    Holmes GL, Ben-Ari Y. The neurobiology and consequences of epilepsy in the developing brain. Pediatric Res 2001;49: 3205.
  • 37
    Lemaire V, Koehl M, Le Moal M, et al. Prenatal stress produces learning deficits associated with an inhibition of neurogenesis in the hippocampus. Proc Natl Acad Sci U S A 2000;97: 110327.
  • 38
    Bartesagfi R, Serrai A. Effects of early environment on granule cell morphology in the dentate gyrus of the guinea-pig. Neuroscience 2001;102: 87100.
  • 39
    Gould E, Beylin A, Tanapat P, et al. Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci 1999;2: 2605.
  • 40
    Schlessinger AR, Cowan WM, Gottlieb DI. An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. J Comp Neurol 1975;159: 14976.
  • 41
    Kehoe P, Shoemaker WJ, Triano L, et al. Adults rats stressed as neonates showed exaggerated behavioral response to both pharmacological and environmental challenges. Behav Neurosci 1998;112: 110.
  • 42
    Kehoe P, Bronzino JD. Neonatal stress alters LTP in freely moving male and female rats. Hippocampus 1999;9: 6518.
  • 43
    Baram TZ, Hatalski CG. Neuropeptide-mediated excitability: a key triggering mechanism for seizure generation in the developing brain. Trends Neurosci 1998;21: 4716.
  • 44
    McCormick CM, Kehoe P, Kovacs S. Corticosterone release in response to repeated short episodes of neonatal isolation: evidence of sensitization. Int J Dev Neurosci 1998;16: 17585.
  • 45
    Stanton ME, Gutierrez YR, Levine S. Maternal deprivation potentiates pituitary-adrenal stress responses in infant rats. Behav Neurosci 1988;102: 692700.
  • 46
    Kehoe P, Mallinson K, McCormick CM, et al. Central allopregnanolone is increased in rat pups in response to repeated, short episodes of neonatal isolation. Dev Brain Res 2000;124: 1336.
  • 47
    Sapolsky RM, Meaney MJ. Maturation of the adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res 1986;39: 6476.
  • 48
    Landfield PW, Waymire J, Lynch G. Hippocampal aging and adrenocorticoids: a quantitative correlation. Science 1978;202: 1098102.
  • 49
    Sapolsky RM. A mechanism of glucocorticoid toxicity in the hippocampus: increased neuronal vulnerability to metabolic insults. J Neurosci 1985;5: 122832.
  • 50
    Sapolsky RM, Krey LC, McEwen BS. Prolonged glucocorticoid exposure reduces hippocampal neuron number: implication for aging. J Neurosci 1985;5: 12216.
  • 51
    Landfield PW, Eldridge JC. The glucocorticoid hypothesis of brain aging and neurodegeneration: recent modification. Acta Endocrinol 1991;125: 5464.
  • 52
    Sapolsky RM. Stress, the aging brain and the mechanisms of neuron death. Cambridge, MA: MIT Press, 1992:423.
  • 53
    Milner B, Squire LR, Kandel ER. Cognitive neuroscience and the study of memory. Neuron 1998;20: 4658.
  • 54
    Silva AJ, Steven CF, Tonegawa S, et al. Deficient hippocampal long-term potentiation in α-calcium-calmodulin kinase II mutant mice. Science 1992;257: 2016.
  • 55
    Mathern GW, Babb TL, Vickrey BG, et al. The clinical-pathologic mechanisms of hippocampal neuron loss and surgical outcomes in temporal lobe epilepsy. Brain 1995;118: 10518.
  • 56
    Nelson KG, Ellenberg JH. Predictors of epilepsy in children who have experienced febrile seizures. N Engl J Med 1976;295: 102933.
  • 57
    Mathern GW, Price G, Rosales C, et al. Anoxia during kainate status epilepticus shortens behavioral convulsions but generates hippocampal neuron loss and supragranular mossy fiber sprouting. Epilepsy Res 1998;30: 13351.
  • 58
    Katzir H, Mendoza D, Mathern GW. Effect of theophylline and trimethobenzamide when given during kainate-induced status epilepticus: an improved histopathologic rat model of human hippocampal sclerosis. Epilepsia 2000;41: 13909.