Seizures In Neonatal Rats Research Articles

Altered hippocampal dendritic spine maturation after hypoxia-induced seizures in neonatal rats

Cognitive comorbidities often follow early-life seizures (ELS), especially in the setting of autism and other neurodevelopmental syndromes. However, there is an incomplete understanding of whether neuronal and synaptic development are concomitantly dysregulated. We have previously shown that hypoxia-induced seizures (HS) in postnatal day (P)10 rats increase acute and later-life hippocampal glutamatergic neurotransmission and spontaneous recurrent seizures, and impair cognition and behavior. As dendritic spines critically regulate synaptic function, we hypothesized that ELS can induce developmentally specific changes in dendritic spine maturation. At intervals during one month following HS in P10 rats, we assessed dendritic spine development on pyramidal neurons in the stratum radiatum of hippocampal area CA1. Compared to control rats in which spine density significantly decreased from P10 to early adulthood (P38), post-seizure rats failed to show a developmental decrease in spine density, and spines from P38 post-seizure rats appeared more immature-shaped (long, thin). In addition, compared to P38 control rats, post-seizure P38 rats expressed significantly more synaptic PSD-95, a marker of mature synapses. These changes were preceded by a transient increase in hippocampal expression of cofilin phosphorylated at Ser3, representing a decrease in cofilin activity. These results suggest that early-life seizures may impair normal dendritic spine maturation and pruning in CA1 during development, resulting in an excess of less efficient synapses, via activity-dependent modification of actin-regulating proteins such as cofilin. Given that multiple neurodevelopmental disorders show similar failures in developmental spine pruning, the current findings may represent a deficit in structural plasticity that could be a component of a mechanism leading to later-life cognitive consequences associated with early-life seizures.

Pre- and post-exposure talampanel (GYKI 53773) against kainic acid seizures in neonatal rats.

AMPA receptors play an important role in the neurobiology of neonatal epilepsy. The present study evaluated the effect of talampanel, a potent and selective non-competitive antagonist of AMPA receptors, against kainic acid-induced continuous seizures (status epilepticus) and other behavioral abnormalities in neonatal rats. Kainic acid was administered at doses of 2 or 4mg/kg, ip to induce seizures and status epilepticus in postnatal 7 days old rat neonates in pre- and post-exposure studies, respectively. Intraperitoneal administration of kainic acid (2 or 4mg/kg) resulted in forelimb/hind-limb scratching defined as automatism, continuous generalized tonic-clonic seizures with loss of righting reflex suggesting status epilepticus and tonic extension. Pre-exposure of talampanel (2.5-10mg/kg, ip) 30min before kainic acid did not affect the onset of kainic acid convulsions. Talampanel at 20mg/kg, ip delayed the commencement of tonic extension, but not status-induced by kainic acid. In contrast, talampanel (5 and 10mg/kg, ip) when administered 5min after kainic acid (4mg/kg, ip) postponed the onset of status epilepticus and tonic extension compared to vehicle treated group. Furthermore, talampanel (10mg/kg, ip) but not GYKI 52466 (20 or 50mg/kg, ip; a non-competitive AMPA/kainate receptor antagonist) stopped the ongoing status epilepticus when administered 10min after the administration of kainic acid. However, seizures re-occurred after 35.98±2.36min. The present results suggested that talampanel is protective in kainic acid-induced neonatal status epilepticus model; however, the time of administration is a crucial factor in determining its effectiveness.

Memantine delayed N-methyl-D-aspartate -induced convulsions in neonatal rats.

Memantine (1-amino-3,5-dimethyladamantane) is a moderate-affinity uncompetitive antagonist of N-methyl-d-aspartate (NMDA) receptors. In this study, we have explored the effect of memantine against N-methyl-d-aspartate (NMDA)-induced seizures in neonatal rats. Here, we evaluated various behavioral seizure abnormalities in neonatal rats (Sprague-Dawley; postnatal day 9) after an intraperitoneal administration of NMDA. Further, we explored whether an acute administration of memantine could protect these neonates against different phases of convulsions induced by NMDA. In a separate study, we have compared the effect of levetiracetam in the same animal model. Exogenous administration of NMDA (30mg/kg., i.p.) in neonatal rats resulted in arrest of activity, emprosthotonos curvature (trunk is bent forward by the entire muscles), myoclonic jerks, and forelimb/hindlimb clonus. The clonus phase in neonates was followed by loss of righting reflex and continuous seizures (for more than 5min) suggesting status epilepticus, tonic extension, and death. Pretreatment of memantine hydrochloride (10-30mg/kg., i.p.) dose-dependently delayed the onset of different phases of convulsions induced by NMDA. Memantine at the highest dose was found to be ataxic in rat neonates, while lower doses were free of any observed behavioral signs of toxicity. Levetiracetam (25mg/kg., i.p.) when administered 30min before the NMDA challenge blocked only the jerk phase and did not affect other phases of NMDA-induced convulsions. These data indicated that memantine and other safer uncompetitive NMDA receptor antagonists may be protective in the management of neonatal seizures.

Comparative research of the effect of routine and every other day ketogenic diet on neurobehavioral damages induced by recurrent seizures in neonatal rats

Objective To compare the effect of routine ketogenic diet and every other day ketogenic diet on neurobehavioral damage induced by recurrent seizures in neonatal rats. Methods 48 postnatal day 8 (P8) SD rats were randomly divided into four groups with 12 in each group: the control group(CONT), the recurrent seizure group(RS+ ND), recurrent seizure + routine ketogenic diet group(RS+ KD) and recurrent seizure+ every other day ketogenic diet group(RS+ KOD). The recurrent seizures model was induced by flurothyl at p9 and last for 8 days. After a day of fasting the postnatal 28 day rats were placed on either ordinary or ketogenic diet according to packet design. Plane righting experiment, cliff avoidance test and negative geotaxis test were used to assess the neurobehavioral performance at p35. Results (1) Plane righting experiment: the plane righting time of RS+ ND group ((0.17±0.39)s) was significantly shorter than that of NS+ ND group ((0.67±0.49)s) (P 0.05). (2) Cliff avoidance test: the cliff avoidance time of RS+ ND group ((12.58±4.83)s) was significantly longer than that of NS+ ND group ((1.92±0.90)s), RS+ KD group((3.33±1.50)s) and RS+ KOD group (P 0.05). Conclusion The ketogenic diet can improve neurobehavioral damage caused by flurothyl-induced recurrent seizures in neonatal rats. The every other day KD group shows a weak intervention effect comparing with the routine KD group. Key words: Neonatal; Seizures; Ketogenic diet; Neurobehavior

Downregulation of Hippocampal GABA after Hypoxia-Induced Seizures in Neonatal Rats

This study aims to determine the expression of Gamma-aminobutyric acid (GABA) following hypoxia in neonatal rats and explore how it may increase susceptibility to epilepsy later in life. A modified model of neonatal hypoxia-induced epileptic susceptibility was simulated by 17 min of hypoxia (5% O(2) and 95% N(2)) in postnatal day (P) 10 rats. Hippocampal glutamate decarboxylase (GAD) and parvalbumin (PV) during the development with or without hypoxia were examined using immunohistochemistry. No detectable neuronal loss was observed in the hippocampus either immediately or 14 days after hypoxia. During the development GAD- and PV-immunoreactivity increased substantially during P 11-13 and reached mature expression in the control rats, and decreased significantly at different time points except for a transient increase during P 11-13 in the hypoxic groups. Our study indicates that downregulation of hippocampal GABA after hypoxia-induced seizures in neonatal rats may contribute to higher epileptic susceptibility in later life.

Morphological and behavioral consequences of recurrent seizures in neonatal rats are associated with glucocorticoid levels

It is well documented that epilepsy can increase neurogenesis in certain brain regions and cause behavioral alternations in patients and different epileptic animal models. A series of experimental studies have demonstrated that neurogenesis is regulated by various factors including glucocorticoid (CORT), which can reduce neurogenesis. Most of studies in animal have been focused on adulthood stage, while the effect of recurrent seizures to immature brain in neonatal period has not been well established. This study was designed to investigate how the recurrent seizures occurred in the neonatal period affected the immature brain and how CORT regulated neurogenesis in immature animals. Neonatal rats were subjected to 3 pilocarpine-induced seizures from postnatal day 1 to day 7. Then neurogenesis at different postnatal ages (i.e. P8, P12, P22, P50) was observed. Behavioral performance was tested when the rats were mature (P40), and plasma CORT levels following recurrent seizures were simultaneously monitored. Rats with neonatal seizures had a significant reduction in the number of Bromodeoxyuridine (BrdU) labeled cells in the dentate gyrus compared with the control groups when the animals were euthanized on P8 or P12 (P<0.05); whereas there was no difference between the two groups on P22. Until P50, rats with neonatal seizures had increased number of BrdU-labeled cells compared with the control group (P<0.05). In Morris water maze task, pilocarpine-treated rats were significantly slower than the control rats at the first and second day, and there were no differences at other days. In probe trial, there was no significant difference in time spent in the goal quadrant between the two groups. Endocrine studies showed a correlation between the number of BrdU positive cells and the CORT level. Sustained increase in circulating CORT levels was observed following neonatal seizures on P8 and P12. Neonatal recurrent seizures can biphasely modulate neurogenesis over different time windows with a down-regulation at early time and up-regulation afterwards, cause persistent deficits in cognitive functions of adults, and increase the circulating CORT levels. CORT levels are related with the morphological and behavioral consequences of recurrent seizures.

Consequences of pilocarpine-induced recurrent seizures in neonatal rats

Accumulated evidence have shown that a series of morphological alternations occur in patients with epilepsy and in different epileptic animal models. Given most of animal model studies have been focused on adulthood stage, the effect of recurrent seizures to immature brain in neonatal period has not been well established. This study was designed to observe the certain morphological changes following recurrent seizures occurred in the neonatal rats. For seizure induction, neonatal Wistar rats were intraperitoneally injected with pilocarpine on postnatal day 1 (P1), P4 and P7. Rat pups were grouped and sacrificed at 1d, 7d, 14d and 42d after the last pilocarpine injection respectively. Bromodeoxyuridine (BrdU) was intraperitoneally administered 36 h before the rats were sacrificed. BrdU single and double labeling with neuronal markers were used to analyze cell proliferation and differentiation. Nissl and Timm staining were performed to evaluate cell loss and mossy fiber sprouting. Rats with neonatal seizures had a significant reduction in the number of Bromodeoxyuridine-(BrdU) labeled cells in the dentate gyrus compared with the control groups when the animals were killed either 1 or 7 days after the third seizure ( P < 0.05) but there was no difference between two groups on P21. On the contrary, BrdU-labeled cells significantly increased in the experimental group compared with control group on P49 ( P < 0.05). The majority of the BrdU-labeled cells colocalized with neuronal marker-NF200 (Neurofilament-200). Nissl staining showed that there was no obvious neuronal loss after seizure induction over all different time points. Rats with the survival time of 42 days after neonatal seizures developed to increased mossy fiber sprouting in both the CA3 region and supragranular zone of the dentate gyrus compared with the control groups ( P < 0.05). Taken together, the present findings suggest that synaptic reorganization only occurs at the later time point following recurrent seizures in neonatal rats, and neonatal recurrent seizures can modulate neurogenesis oppositely over different time window with a down-regulation at early time and up-regulation afterwards.

Neonatal Seizures: Is a Novel, Mechanism-Based Treatment Finally on the Horizon?

NKCC1 Transporter Facilitates Seizures in the Developing Brain Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE, Staley KJ Nat Med 2005;11(11):1205–1213 During development, activation of Cl–-permeable GABAA receptors (GABAA-R) excites neurons as a result of elevated intracellular Cl– levels and a depolarized Cl– equilibrium potential (ECl). GABA becomes inhibitory as net outward neuronal transport of Cl– develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na+-K+-2Cl– cotransporter (NKCC1) facilitates the accumulation of Cl– in neurons. The NKCC1 blocker bumetanide shifted ECl negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABAA-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl–-extruding transporter (KCC2) in human and rat cortex showed that Cl– transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures. Depolarizing GABA Acts on Intrinsically Bursting Pyramidal Neurons to Drive Giant Depolarizing Potentials in the Immature Hippocampus Sipilä ST, Huttu K, Soltesz I, Voipio J, Kaila K J Neurosci 2005;25(22):5280–5289 Spontaneous periodic network events are a characteristic feature of developing neuronal networks, and they are thought to play a crucial role in the maturation of neuronal circuits. In the immature hippocampus, these types of events are seen in intracellular recordings as giant depolarizing potentials (GDPs) during the stage of neuronal development when GABAA-mediated transmission is depolarizing. However, the precise mechanism how GABAergic transmission promotes GDP occurrence is not known. Using whole-cell, cell-attached, perforated-patch, and field-potential recordings in hippocampal slices, we demonstrate here that CA3 pyramidal neurons in the newborn rat generate intrinsic bursts when depolarized. Furthermore, the characteristic rhythmicity of GDP generation is not based on a temporally patterned output of the GABAergic interneuronal network. However, GABAergic depolarization plays a key role in promoting voltage-dependent, intrinsic pyramidal bursting activity. The present data indicate that glutamatergic CA3 neurons have an instructive, pacemaker role in the generation of GDPs, whereas both synaptic and tonic depolarizing GABAergic mechanisms exert a temporally nonpatterned, facilitatory action in the generation of these network events.

NKCC1 transporter facilitates seizures in the developing brain

During development, activation of Cl(-)-permeable GABA(A) receptors (GABA(A)-R) excites neurons as a result of elevated intracellular Cl(-) levels and a depolarized Cl(-) equilibrium potential (E(Cl)). GABA becomes inhibitory as net outward neuronal transport of Cl(-) develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) facilitates the accumulation of Cl(-) in neurons. The NKCC1 blocker bumetanide shifted E(Cl) negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABA(A)-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl(-)-extruding transporter (KCC2) in human and rat cortex showed that Cl(-) transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.

Long-term effects of seizures in neonatal rats on spatial learning ability and N-methyl- d-aspartate receptor expression in the brain

For the purpose of investigating the long-term effects of seizures in neonatal rats on spatial learning ability and N-methyl- d-aspartate (NMDA) receptor expression in adult rat brain, a seizure was induced by inhalant flurothyl daily in neonatal Wistar rats from postnatal day 6 (P6). The authors assigned six rats each averagely into the single-seizure group, the recurrent-seizure group (seizures induced in six consecutive days), and the control group. During P60 to P65, the rats were tested for spatial learning ability with the Morris water maze task. On P75, the authors examined protein expression of the NMDA receptor (NR) subunits, NR1, 2A, 2B, 2C, and 2D, in the cerebral cortex and hippocampus by Western blotting analysis. On P65, the escape latencies from the water maze of the rats in the recurrent-seizure group were significantly longer than those of the control rats, but there was no difference between the single-seizure group and the control group. NR subunit expression in the cerebral cortex and hippocampus of the rats with single seizure was similar to those in the control rats. Compared with the control rats, the protein expressions of NR1, NR2A and NR2B in the cerebral cortex and NR2A in the hippocampus of the recurrent-seizure group was significantly decreased, but NR2C protein expression in the cerebral cortex and hippocampus significantly increased. Recurrent seizures induced in neonatal rats might cause long-term spatial learning ability deficit and modify NR expression in the cerebral cortex and hippocampus of adult rats. The results suggest that abnormal NR expression might play an important role in long-term spatial learning ability deficit induced by recurrent seizures in early life.

Acute effects of MK801 on kainic acid-induced seizures in neonatal rats.

Kainic acid (KA) causes behavioral and electrographic status epilepticus (SE) in rats of all ages. In adult rats, the noncompetitive N-methyl-D-aspartate (NMDA) channel blocker MK801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine ) is anticonvulsant against KA-induced seizures: it reduces their severity and protects against neuronal damage, although it may worsen electrographic seizures. Here we examined the effects of MK801 on KA seizures in the immature brain. Neonatal rats (P11-P12) were pretreated with MK801 (0.01, 0.1, 0.5 or 1.0 mg/kg, i.p.) or saline twenty minutes prior to KA (2 mg/kg, i.p.). Clinical seizure behavior was monitored for > 6 hrs, and in some rats the EEG was monitored with an intrahippocampal or intracortical electrode. MK801 caused immobility alternating with hyperactivity, ataxia, scratching and sometimes alternate limb cycling, which correlated with the appearance of spikes on the EEG. Compared to KA alone or KA preceded by 0.01 mg/kg MK801, the higher doses of MK801 (0.1, 0.5 and 1.0 mg/kg) significantly lowered the latency to electrographic seizures (P < 0.001), ictal scratching (P < 0.0001), and status epilepticus (P < 0.0001). MK801 pretreatment did not lower significantly the death rate due to KA seizures. No histologic damage was seen after MK801, KA or both agents together. These results suggest that MK801 exacerbates KA-induced seizures in the neonatal brain, and may even cause ictal behavioral and electrographic manifestations by itself. The findings point to an age-dependency of NMDA antagonist action, and suggest caution in considering the use of NMDA antagonists in neonates.

Benzodiazepines and the developing rat: A critical review

This paper reviews: (1) the development of benzodiazepine binding-sites and the GABA system; (2) the evidence that prenatal exposure to benzodiazepines can cause malformations; (3) other persisting effects of developmental exposure to benzodiazepines; and (4) the behavioral effects of benzodiazepines (and other relevant drugs) in immature animals. The review concentrates on the rat, since fundamental work in other species is scarce. The data on neurochemical development are found to be generally consistent; however, reports that the enhancement of benzodiazepine binding by GABA varies with age are controversial. The physical development of the rat is disturbed only by extremely high doses of benzodiazepines. The evidence for persisting effects after early exposure to benzodiazepines is impressive at first sight, but in most studies, confounding variables have not been eliminated. Startle and some learning tasks are affected by prenatal diazepam; submissiveness is affected by neonatal lorazepam; social behaviour and convulsions are affected by neonatal CGS 8216. Benzodiazepines inhibit chemically-induced seizures in neonatal rats, but the developmental profile of sensitivity to the convulsants is disputed. Benzodiazepines stimulate motor behavior in the neonatal rat.

Behavioral and electrographic patterns induced by systemic administration of kainic acid in developing rats.

Abstract Behavioral and electrographic changes induced by systemic injection of 3–6 mg/kg of kainic acid (KA) have been studied during development in 1- to 28-day-old rats. In the first week of life, a few minutes after the administration of the toxin, animals developed motor signs such as scratching movements, hypo- and hyperactivity, loss of postural control, tremors and hyperextension of the limbs. No changes of the electrical cortical and hippocampal activity were detected. Similar, but more accentuated motor signs occur when KA was injected during the second week of postnatal life. In this case, on the electroencephalogram (EEG), bursts of spikes or polispikes localized in the cortex appeared. These bursts became more and more frequent and after only 20 min spread to the hippocampus. No correlation between the electrical and behavioral changes were observed. By the third week of life, in addition to motor signs, systemic kainic acid induced wet dog shakes (WDS) and limbic seizures. These were characterized by rearing, loss of postural control, masticatory movements and forepaw tremors. The EEG was characterized by primary hippocampal discharges that may spread to the cortex. Usually the limbic seizures become more and more frequent, leading to a status epilepticus. In another group of experiments, higher doses of KA (6–12 mg/kg) have been injected in curarized and artificially respirated animals of the same age. During the first days of life (1–7), unlike in the corresponding group of freely moving animals, KA induced sequences of slow spikes localized in the cortex and after 10–20 min spread to the hippocampus. In the following age-groups no differences between the curarized and free moving animals were detected. The different sensitivity of systemic KA to induce limbic seizures in neonatal rats may be related to the immaturity of pre- and post-synaptic glutamergic receptors in the hippocampus. Moreover, a different degree of maturity of cortical versus hippocampal glutamergic receptors may account for the preferential activation of the cortex versus hippocampus during the first period of life.