Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy

doi:10.1016/S0306-4522(01)00327-X

Neuroscience

Volume 107, Issue 1, 9 November 2001, Pages 71-84

https://doi.org/10.1016/S0306-4522(01)00327-X Get rights and content

Abstract

Studies were undertaken to examine the effects recurrent early-life seizures have on the ability of rats to acquire spatial memories in adulthood. A minute quantity of tetanus toxin was injected unilaterally into the hippocampus on postnatal day 10. Within 48 h, rats developed recurrent seizures that persisted for 1 week. Between postnatal days 57 and 61, rats were trained in a Morris water maze. Toxin-injected rats were markedly deficient in learning this task. While these rats showed gradual improvement in escape latencies over 20 trials, their performance always lagged behind that of controls. Poor performance could not be explained by motor impairments or motivational difficulties since swimming speed was similar for the groups. Only eight of 16 toxin-injected animals showed focal interictal spikes in the hippocampus during electroencephalographic recordings. This suggests that learning deficiencies and chronic epilepsy may be independent products of recurrent early-life seizures. A quantitative analysis of hippocampus revealed a significant decrease in neuronal density in stratum pyramidale of experimental rats. However, the differences were largely explained by a concomitant increase in the area of stratum pyramidale. Studies of glial fibrillary acidic protein expression and spread of horseradish peroxidase-conjugated tetanus toxin in the hippocampus suggest that the dispersion of cell bodies in stratum pyramidale can neither be explained by a reactive gliosis nor the direct action of the toxin itself.

Taken together, we suggest that recurrent seizures beginning in early life can lead to a significant deficiency in spatial learning without ongoing hippocampal synchronized network discharging or a substantial loss of hippocampal pyramidal cells.

Section snippets

Intrahippocampal injection of tetanus toxin

Ten-day-old (PND 10) Wistar rat pups (Harlan) were anesthetized with an i.p. injection of ketamine/xylazine mixture (30 mg/1.5 mg per kg, respectively) and placed in a modified neonatal stereotaxic head holder (Snead and Stephens, 1982). Animals were kept warm with a thermally regulated metal plate. In 36 animals, a minute quantity (2 ng in 16 nl) of tetanus toxin was stereotaxically injected into the CA3 subfield of the right hippocampus at a rate of 4 nl per min. Stereotaxic coordinates for

Tetanus toxin-induced seizures in infant rats

Tetanus toxin-treated rat pups exhibited wild running seizures within 48–72 h of injection. The onset of wild running episodes frequently started when an animal underwent a series of prolonged wet dog shakes. The running phase of the seizure followed immediately and was interrupted frequently by episodes of intense fore limb clonus, repetitive chewing and licking and eye and facial movements. Pups would also vocalize during the later phase of the seizure episode. Seizures ended with either a

Discussion

Mental retardation is not an inevitable consequence of childhood epilepsy. However, subtle impairments in intellectual functioning have been reported in many individuals with a history of seizures in early life (Cassidy and Corbett, 1997). Furthermore, the majority of children who suffer from severe epileptic syndrome, such as infantile spasm and Lennox Gastaut syndrome, have significant learning disabilities and mental retardation. Clinical cases of children with such difficult to control

Acknowledgements

This work was supported by NIH Grants NS18309, NS37171, a Mental Retardation Research Center Grant HD24064 and a grant from the Epilepsy Foundation of America.

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Tetanus toxin provides a reliable means of inducing recurrent spontaneous epileptic seizures in the hippocampus and neocortex. The hippocampal tetanus toxin model differs from other methods of inducing spontaneous limbic seizures in that it does not require status epilepticus, and it does not have major cell loss as an early component of the syndrome. Indeed, it has proved useful in showing that seizures can occur in the absence of gross pathology, and that long-term behavioral complications of seizures need not depend on neuronal loss, or ongoing epileptic activity.
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Past studies have demonstrated that inducing several seizures or continuous seizures in neonatal or adult rats results in impairments in learning and memory. The impact of a single acute seizure on learning and memory has not been investigated in mice. In this study, we exposed adult 129SvEvTac mice to the inhalant flurothyl until a behavioral seizure was induced. Our study consisted of 4 experiments where we examined the effect of one seizure before or after delay fear conditioning. We also included a separate cohort of animals that was tested in the open field after a seizure to rule out changes in locomotor activity influencing the results of memory tests. Mice that had experienced a single seizure 1 h, but not 6 h, prior to training showed a significant impairment in associative conditioning to the conditioned stimulus when compared with controls 24 h later. There were no differences in freezing one day later for animals that experienced a single seizure 1 h after associative learning. We also found that an acute seizure reduced activity levels in an open-field test 2 h but not 24 h later. These findings suggest that an acute seizure occurring immediately before learning can have an effect on the recall of events occurring shortly after that seizure. In contrast, an acute seizure occurring shortly after learning appears to have little or no effect on long-term memory. These findings have implications for understanding the acute effects of seizures on the acquisition of new knowledge.
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Neonatal seizures caused by perinatal asphyxia and hypoxic–ischemic encephalopathy can be refractory to conventional anticonvulsants. This may be due to the depolarizing effects of gamma-aminobutyric acid (GABA) achieved by the activity of the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC1). The aim of this study is to evaluate the long-term effects of bumetanide, a NKCC1 inhibitor, on hippocampal neurogenesis and seizure susceptibility in hypoxia-induced neonatal seizure model. Wistar rats were subjected to hypoxia-induced neonatal seizures at postnatal day 10 (P10). Following acute seizures, the rats were treated with intraperitoneal injection (i.p.) of bumetanide at a dose of 0.5 mg/kg for 3 weeks. In later adulthood, hypoxia-induced seizures increased the number of newborn dentate gyrus cells (DGCs), promoted mossy fiber sprouting (MFS) and reduced the apical dendritic complexity of newborn DGCs 1 month after the insults. In addition, these seizures resulted in long-lasting consequences, such as spontaneous electroencephalography (EEG) seizures, though spatial learning impairments were not seen. Bumetanide treatments significantly enhanced cell proliferation and dendritic development of newborn DGCs after neonatal seizures, accompanied by the decreased seizure activity. However, systemic administration of bumetanide resulted in much lower brain concentrations, and was incompatible with NKCC1 inhibition in blood–brain barrier (BBB)-protected brain tissue. Our results suggested that bumetanide might have long-term effects in suppressing seizure activity, and altering the neurogenesis after neonatal seizures. These effects of bumetanide may be mediated by the targets outside the BBB-protected central nerve system (CNS) or CNS-located target(s) other than NKCC1.
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Seizures are more common in the neonatal period than any other time in life (Cowan, 2002) and both experimental evidence (Cornejo et al., 2008; Holmes et al., 1998; Isaeva et al., 2009; Jensen et al., 1992; Kleen et al., 2011; Lee et al., 2001) and clinical evidence suggest that early life seizures negatively impact later neurodevelopment independently of the underlying pathology (Glass et al., 2009; McBride et al., 2000; Miller et al., 2002; van der Heide et al., 2012).
Hypoxemic events are common in sick preterm and term infants and represent the most common cause of seizures in the newborn period. Neonatal seizures often lack clinical correlates and are only recognized by electroencephalogram (EEG). The mechanisms leading from a hypoxic/ischemic insult to acute seizures in neonates remain poorly understood. Further, the effects of hypoxia on EEG at various developmental stages have not been fully characterized in neonatal animals, in part due to technical challenges. We evaluated the impact of hypoxia on neonatal mouse EEG to define periods of increased susceptibility to seizures during postnatal development. Hippocampal and cortical electrodes were implanted stereotaxically in C57BL/6 mice from postnatal age 3 (P3) to P15. Following recovery, EEG recordings were obtained during baseline, acute hypoxia (4% FiO₂ for 4 min) and reoxygenation. In baseline recordings, maturation of EEG was characterized by the appearance of a more continuous background pattern that replaced alternating high and low amplitude activity. Clinical seizures during hypoxia were observed more frequently in younger animals (100% P3–4, 87.5% P5–6, 93% P7–8, 83% P9–10, 33% P11–12, 17% P15, r² = 0.81) and also occurred at higher FiO₂ in younger animals (11.2 ± 1.1% P3–P6 vs. 8.9 ± 0.8% P7–12, p < 0.05). Background attenuation followed the initial hypoxemic seizure; progressive return to baseline during reoxygenation was observed in survivors. Electrographic seizures without clinical manifestations were observed during reoxygenation, again more commonly in younger animals (83% P3–4, 86% P5–6, 75% P7–8, 71% P9–10, 20% P11–12, r² = 0.82). All P15 animals died with this duration and degree of hypoxia. Post-ictal abnormalities included burst attenuation and post-anoxic myoclonus and were more commonly seen in older animals. In summary, neonatal mice exposed to brief and severe hypoxia followed by rapid reoxygenation reliably develop seizures and the response to hypoxia varies with postnatal age and maturation.

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Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy

Abstract

Section snippets

Intrahippocampal injection of tetanus toxin

Tetanus toxin-induced seizures in infant rats

Discussion

Acknowledgements

Dev. Brain Res.

Epilepsy Res.

Neuroscience

Neuropharmacology

Neurosci. Lett.

Neuropharmacology

Seizure

Neuroscience

Brain Res.

Behav. Brain Res.

Brain Dev.

Dev. Brain Res.

Brain Res. Dev. Brain Res.

Behav. Brain Res.

Brain Res.

Epilepsia

Epilepsy Res.

Trends Biochem. Sci.

Neuroscience

Brain Res. Dev. Brain Res.

Neurobiol. Learning Memory

Neuroscience

Dev. Med. Child Neurol.

Dev. Brain Res.

Damage limited to the hippocampal region produces long-lasting memory impairment in monkeys

J. Neurosci.

Neuronal loss induced in limbic pathways by kindling: evidence for induction of hippocampal sclerosis by repeated brief seizures

J. Neurosci.

Hyperglycemia increases neurological damage and behavioral deficits from posttraumatic secondary ischemic insults

J. Neurotrauma