The Mechanisms of Stress in Epileptogenesis

Cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) catalyzes the initial step in the biosynthesis of neurosteroids within the brain. We sought to determine which cells express P450cc and whether neurosteroids play a role in the regulation of epileptogenesis following pilocarpine-induced status epilepticus (SE). Rats experienced uninterrupted SE or SE terminated with diazepam at 60, 120, and 180 min. P450scc induction in CA3 hippocampus was determined by double immunolabeling with P450scc antiserum and monoclonal antibodies against GFAP (astrocytes), RIP (oligodendrocytes), or heme oxygenase-1 (microglia). SE was associated with P450scc induction in many astrocytes and a small number of microglia and oligodendrocytes in the hippocampal CA3 strata radiatum and lacunosum-moleculare. The extent of P450scc induction increased with increasing SE duration. Paradoxically, increased P450scc induction in rats experiencing SE for 180 min or more was associated with the delayed onset of spontaneous recurrent seizures. Treatment with the 5 alpha-reductase inhibitor finasteride (100 mg/kg/day for 25 days), which inhibits the synthesis of gamma-aminobutyric acid (GABA)(A) receptor modulating neurosteroids such as allopregnanolone, was associated with a significant reduction in time to the onset of spontaneous seizures in rats exposed to 180-min but not 90-min SE. P450scc is induced by SE in a diverse population of hippocampal glia. Induction of P450scc is associated with the delayed onset of spontaneous seizures. Conversely, inhibition of neurosteroid synthesis accelerated the onset of spontaneous seizures, but only in animals exhibiting significant increases in P450scc. These findings suggest that induction of neurosteroid synthesis in reactive glial cells is associated with delayed onset of spontaneously recurrent seizures.

The "silent period" is a characteristic of human localization-related symptomatic epilepsy. In mesial temporal lobe epilepsy (MTLE), it follows an initial precipitating injury, and in animal models of MTLE in which brain damage is artificially created, there is also a prolonged interval between injury and the onset of spontaneous seizures. The neuronal reorganization responsible for epileptogenesis presumably takes place during this silent interval; however, the functional correlates of this process are poorly understood. We have previously described high-frequency (250 to 500 Hz) oscillations, called fast ripples (FR), in the hippocampus and entorhinal cortex (EC) of intrahippocampal kainic acid (KA)-injected rats and patients with MTLE that are confined to the region of spontaneous seizure generation. We have proposed, therefore, that FR reflect the mechanisms responsible for epileptogenesis. If this is the case, they should appear during the process of epileptogenesis, before the appearance of spontaneous seizures. The purpose of the present study was to record continuously from rats after KA injection to compare the temporal development of FR with spontaneous seizures. Additional goals were to determine in these rats after spontaneous seizures begin (a) the volume of tissue in which FR can be recorded in hippocampus and EC, (b) the multiple-unit and field potential correlates of FR oscillations, and (c) whether there is an association of FR with mossy fiber sprouting. After unilateral KA injection in the posterior hippocampus, interictal field epileptic activity and single-unit activity were recorded from freely moving animals using multiple-contact microelectrodes in dentate gyrus (DG) and EC. One group of animals underwent continuous recording to determine the time of onset of both FR oscillations and spontaneous seizures. A second group was implanted after behavioral seizures began to measure the area within which FR could be recorded as well as their unit and field potential correlates. The neo-Timm method was used to reveal mossy fiber sprouting, and gray value analysis was used to measure the intensity of sprouting in the inner molecular layer of DG. In KA-injected rats, FR were observed in hippocampal areas adjacent to the lesion and in the ipsilateral EC 11 to 14 days after injection, whereas spontaneous behavioral seizures occurred 2 to 4 months after injection. Analysis of depth profiles of interictal FR in the DG and EC showed that they were generated in local areas with a volume of about 1.0 mm3, and unit recordings indicated that they reflected fields of hypersynchronous action potentials. FR were found in areas of DG with more intensive mossy fiber sprouting. However, the correspondence was not absolute. The electrophysiological and anatomical data are consistent with the participation of FR oscillations, within small neuronal assemblies, in the development of chronic epileptogenesis. It is hypothesized that small clusters of pathologically interconnected neurons develop after focal hippocampal injury and that these clusters are capable of generating powerful hypersynchronous bursts of action potentials, which initiate epileptogenesis via a kindling effect. As the silent period progresses, a network of such clusters is formed that allows the development of discharges that spread throughout the limbic system. When this network engages brain areas that control motor activity, clinical seizures occur and the silent period ends.

Objective SCN8A epileptic encephalopathy is caused predominantly by de novo gain‐of‐function mutations in the voltage‐gated sodium channel Nav1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti‐seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy.MethodsUsing immunohistochemistry, we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase and Nav1.6, and Kir4.1 channel currents were assessed in astrocytes in wild‐type (WT) mice and mice carrying the N1768D SCN8A mutation (D/+).ResultsAstrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium‐sensitive Kir4.1 currents compared to age‐matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures.SignificanceAstrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced Kir4.1‐mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression.

Epilepsy therapy is based on drugs that treat the symptoms rather than the underlying mechanisms of the disease (epileptogenesis). There are no treatments for preventing seizures or improving disease prognosis, including neurological comorbidities. The search of pathogenic mechanisms of epileptogenesis highlighted that neuroinflammatory cytokines [i.e. interleukin-1β (IL-1β), tumour necrosis factor-α (Tnf-α)] are induced in human and experimental epilepsies, and contribute to seizure generation in animal models. A major role in controlling the inflammatory response is played by specialized pro-resolving lipid mediators acting on specific G-protein coupled receptors. Of note, the role that these pathways have in epileptogenic tissue remains largely unexplored. Using a murine model of epilepsy, we show that specialized pro-resolving mechanisms are activated by status epilepticus before the onset of spontaneous seizures, but with a marked delay as compared to the neuroinflammatory response. This was assessed by measuring the time course of mRNA levels of 5-lipoxygenase (Alox5) and 15-lipoxygenase (Alox15), the key biosynthetic enzymes of pro-resolving lipid mediators, versus Il1b and Tnfa transcripts and proteins. In the same hippocampal tissue, we found a similar delayed expression of two main pro-resolving receptors, the lipoxin A4 receptor/formyl peptide receptor 2 and the chemerin receptor. These receptors were also induced in the human hippocampus after status epilepticus and in patients with temporal lobe epilepsy. This evidence supports the hypothesis that the neuroinflammatory response is sustained by a failure to engage pro-resolving mechanisms during epileptogenesis. Lipidomic LC-MS/MS analysis showed that lipid mediator levels apt to resolve the neuroinflammatory response were also significantly altered in the hippocampus during epileptogenesis with a shift in the biosynthesis of several pro-resolving mediator families including the n-3 docosapentaenoic acid (DPA)-derived protectin D1. Of note, intracerebroventricular injection of this mediator during epileptogenesis in mice dose-dependently reduced the hippocampal expression of both Il1b and Tnfa mRNAs. This effect was associated with marked improvement in mouse weight recovery and rescue of cognitive deficit in the novel object recognition test. Notably, the frequency of spontaneous seizures was drastically reduced by 2-fold on average and the average seizure duration was shortened by 40% after treatment discontinuation. As a result, the total time spent in seizures was reduced by 3-fold in mice treated with n-3 DPA-derived protectin D1. Taken together, the present findings demonstrate that epilepsy is characterized by an inadequate engagement of resolution pathways. Boosting endogenous resolution responses significantly improved disease outcomes, providing novel treatment avenues.

Temporal lobe epilepsy alters adult neurogenesis. Existing experimental evidence is mainly from chronic models induced by an initial prolonged status epilepticus associated with substantial cell death. In these models, neurogenesis increases after status epilepticus. To test whether status epilepticus is necessary for this increase, we examined precursor cell proliferation and neurogenesis after the onset of spontaneous seizures in a model of temporal lobe epilepsy induced by unilateral intrahippocampal injection of tetanus toxin, which does not cause status or, in most cases, detectable neuronal loss. We found a 4.5 times increase in BrdU labeling (estimating precursor cells proliferating during the 2nd week after injection of toxin and surviving at least up to 7days) in dentate gyri of both injected and contralateral hippocampi of epileptic rats. Radiotelemetry revealed that the rats experienced 112±24 seizures, lasting 88±11s each, over a period of 8.6±1.3days from the first electrographic seizure. On the first day of seizures, their duration was a median of 103s, and the median interictal period was 23min, confirming the absence of experimentally defined status epilepticus. The total increase in cell proliferation/survival was due to significant population expansions of: radial glial-like precursor cells (type I; 7.2×), non-radial type II/III neural precursors in the dentate gyrus stem cell niche (5.6×), and doublecortin-expressing neuroblasts (5.1×). We conclude that repeated spontaneous brief temporal lobe seizures are sufficient to promote increased hippocampal neurogenesis in the absence of status epilepticus.

Mislocalization of h channel subunits underlies h channelopathy in temporal lobe epilepsy

In secondary epilepsy, a seizure-prone neural network evolves during the latent period between brain injury and the onset of spontaneous seizures. The nature of the evolution is largely unknown, and even its completeness at the onset of seizures has recently been challenged by measures of gradually decreasing intervals between subsequent seizures. Sequential calcium imaging of neuronal activity, in the pyramidal cell layer of mouse hippocampal in vitro preparations, during early post-traumatic epileptogenesis demonstrated rapid increases in the fraction of neurons that participate in interictal activity. This was followed by more gradual increases in the rate at which individual neurons join each developing seizure, the pairwise correlation of neuronal activities as a function of the distance separating the pair, and network-wide measures of functional connectivity. These data support the continued evolution of synaptic connectivity in epileptic networks beyond the latent period: early seizures occur when recurrent excitatory pathways are largely polysynaptic, while ongoing synaptic remodeling after the onset of epilepsy enhances intranetwork connectivity as well as the onset and spread of seizure activity.

The α2 adrenoreceptor agonist clonidine suppresses evoked and spontaneous seizures, whereas the α2 adrenoreceptor antagonist idazoxan promotes seizures in amygdala-kindled kittens

Neuroinflammatory priming to stress is differentially regulated in male and female rats

The immature brain differs from the adult brain in its susceptibility to seizures, seizure characteristics, and responses to antiepileptic drugs (AEDs) (1). Experimental evidence from animal models has revealed factors that may contribute to the age dependence of epileptic syndromes, and further suggests that early life seizures or treatment with AEDs can alter the normal maturation of brain function (1). To understand fully the age dependence of childhood epilepsies and to optimize treatments, it is necessary to consider maturational differences in the cellular and molecular mechanisms of epilepsy and how these may be functionally altered by early-life seizures or AED treatment. We discuss the developmental regulation of several factors and how these may contribute to seizure susceptibility and epileptogenesis in the developing brain. Seizure incidence is highest in the first year of life, and decreases with age throughout childhood and adolescence (2), and the majority of seizures in the first year of life are symptomatic (i.e., triggered by fever or hypoxia) (3). Early-life seizures can differ qualitatively from seizures in the adult. For instance, the electroencephalogram (EEG) in neonatal seizures frequently shows multifocal and asynchronous epileptiform activity (2), and behavioral seizures may be difficult to distinguish from spontaneous and voluntary movement (4). Although seizures later in childhood may resemble those in the adult, the etiologies of childhood epilepsies are different and can be highly age specific (5). The immature brain can differ from the adult brain in its response to conventional AEDs (6). For example, neonatal seizures can be refractory to AEDs that are potent anticonvulsants in adults (7). Some types of seizure that are generally restricted to early childhood, such as absence or febrile seizures, respond to only a few of the AEDs that are commonly effective in adults (8,9). Children also show greater individual variation in responses and adverse cognitive side effects to AEDs (10). A major concern in neonatal epileptology is whether prolonged AED treatment may impair the normal activity-dependent maturation of brain function. An adverse effect of therapy is supported by a prospective study that revealed that children who were treated with phenobarbital (PB) for febrile seizures exhibited long-term cognitive and intellectual impairment (11,12). The long-term effects of other AEDs have yet to be systematically examined by similar prospective studies in humans. In terms of prognosis, neonatal seizures represent a heterogeneous group (3,13), and it is difficult to identify which clinical characteristics of neonatal seizures determine a poor prognosis. Certain seizures can be benign, as in the case of neonatal hypocalcemic or hypoglycemic seizures in the first week of life (3), or simple febrile seizures in childhood (14). However, other early-life seizures may be related to long-term epilepsy. There is an incidence of later-life epilepsy in cases of symptomatic seizures in the neonate or complex and recurrent febrile seizures in childhood (2,15–18). A controversial question in clinical neonatal and pediatric epileptology is whether early-life seizures per se adversely alter brain maturation, leading to epilepsy or other neurologic syndromes. Patients with temporal lobe epilepsy (TLE) associated with mesial temporal sclerosis (MTS) have a reported increased incidence of previous neonatal or childhood febrile seizures (19–23). Van Landingham et al. (24) found that prolonged febrile convulsions in a small fraction of infants were associated with acute hippocampal edema and delayed hippocampal atrophy. These observations raise the possibility that childhood seizures that occur during a critical maturational period could alter brain development to increase the susceptibility to MTS and TLE. However, prospective and community-based studies have found that childhood febrile seizures are not more likely to be associated with TLE and MTS than are other types of generalized epilepsy (25–27). Thus, although febrile seizures are associated with an increased risk of epilepsy, it is not clear whether the seizures contribute to epileptogenesis or that they are merely indicative of an epilepsy-prone brain. In summary, clinical experience suggests some major principles regarding the relationship between brain maturation and epilepsy. First, the immature brain is more susceptible to seizures compared with the adult brain. Second, the response of the immature brain to AEDs is more variable, and prolonged AED treatment in early life may adversely alter brain development. Third, certain types of seizure in early life are associated with increased likelihood of chronic epilepsy and may themselves contribute to epileptogenesis. Knowledge of age-specific mechanisms of epilepsy and the potential consequences of early-life seizures or AED treatment on brain maturation will help to understand better the age dependence of childhood epilepsies and to optimize age-appropriate treatments. Animal studies indicate that the early postnatal period represents a critical developmental window in which synaptogenesis is ongoing and neuronal plasticity is increased compared with the adult (28–31). Excitatory synaptic transmission mediated by glutamate receptors is required for these processes and is enhanced in the immature brain compared with that of the adult (29,30). Synaptic density undergoes a postnatal overshoot before being "pruned" to adult levels (28,31), and this is paralleled by a relative overexpression of glutamate receptors (32,33). In rat, glutamate-receptor expression is highest in the second postnatal week (32,33), a developmental stage that is roughly analogous to the human term neonate with respect to a number of anatomic, physiologic, and biochemical parameters (34). Additional factors that govern synaptic transmission and neuronal excitability continue to change during this developmental window, including the expression and molecular composition of neurotransmitter receptors and transporters, neuromodulatory peptides and neuropeptide receptors, voltage-gated ion channels, and mechanisms of ionic homeostasis. These factors and their possible relationship to epilepsy in the developing brain are discussed later. Glutamate is the major excitatory neurotransmitter in the brain, and there are several subtypes of glutamate receptor. These include the N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate (KA) subtypes of ionotropic receptors (35), as well as different classes of metabotropic glutamate receptors (36,37). AMPA and KA receptors mediate fast excitatory signaling as they exhibit rapid activation and desensitization, and operate linearly near the resting membrane potential. NMDA receptors play a more modulatory role, as their activation requires concurrent glutamate binding and membrane depolarization and results in slower and longer-lasting excitation. NMDA-receptor channels are highly permeable to Ca2+ (in addition to Na+ and K+), and the influx of Ca2+ through NMDA receptors can trigger signaling pathways that regulate synaptic function and activity-driven synaptogenesis (38,39). The kinetics and permeation properties of NMDA receptors also underlie their key role in pathophysiologic processes such as ictal seizure discharges and hypoxic/ischemic neuronal injury (40–42). Binding studies in the rat indicate that NMDA-receptor density peaks late in the first postnatal week in many forebrain structures, including hippocampus and neocortex (32)(Fig. 1). AMPA-receptor density appears to peak later in the second postnatal week around P10 (32), whereas kainate receptors gradually increase over the first few weeks of life (43). The first 2–3 postnatal weeks represent a window during which glutamate-mediated synaptic plasticity is enhanced (28), and certain brain regions exhibit heightened susceptibility to the epileptogenic and excitotoxic effects of glutamate-receptor agonists (33,44–46). Thus, the overshoot in expression of functional glutamate receptors is likely to play a major role in the increased excitability of the brain in early postnatal development. Schematic depiction of the maturational changes in glutamate and γ-aminobutyric acid (GABA) inotropic receptor expression and function in the developing rat forebrain. Glutamate receptors show an early postnatal overshoot before gradually declining to mature levels. N-methyl-D-aspartate (NMDA)-receptor binding peaks late in the first postnatal week (29), whereas AMPA receptor binding peaks at around P10 (29). Kainate-receptor binding is initially low and shows a gradual increase to adult levels by the fourth postnatal week (40). GABA receptors are expressed early in development (66), but initially mediate depolarizing responses to GABA (25,67). Inhibitory GABA receptor–mediated potentials gradually appear over the first 3 postnatal weeks (67). Thus, early in postnatal life, mechanisms of synaptic excitation predominate over inhibitory mechanisms. The molecular composition of glutamate receptors also is developmentally regulated, and may contribute to the enhanced excitability of the immature brain. The functional properties of glutamate receptors are determined by the particular combination of molecular subunits that compose each receptor (35). In neocortex and hippocampus, expression of the NMDA-receptor subunit NR2B relative to the expression of NR2A is much higher during early postnatal development compared with that in adulthood (47,48). Recombinant NMDA receptors that contain predominantly NR2B exhibit slower decay times than do those that contain predominantly NR2A (48), and the developmental increase in NR2A expression results in a maturational shortening of NMDA receptor–mediated synaptic currents (49). Thus, synaptic excitability and plasticity may be increased in the immature brain by the increased duration of NMDA receptor–mediated excitation and increased Ca2+ influx. Notably, most AMPA and kainate receptors in the mature brain show little relative permeability to Ca2+, but a larger proportion of these channels may exhibit Ca2+ permeability in the immature brain (50). AMPA receptors that lack a GluR2(B) subunit exhibit significantly higher permeability to Ca2+ and other divalent cations than do those that contain a GluR2(B) subunit (51–54), and the ratio of expression of GluR2 subunits to that of other AMPA-receptor subunits appears to be significantly lower in immature neocortex and hippocampus compared with those of the adult (55). This suggests that a larger proportion of AMPA receptors are permeable to Ca2+ in immature neurons in these brain regions, and may therefore mediate pathophysiologic events in early postnatal life, similar to NMDA receptors (50,56). Consistent with this notion, transient knockdown of GluR2 expression using hippocampal microinfusions of antisense mRNA was recently shown to cause spontaneous seizure-like behaviors in young rats (57). There are at least eight cloned metabotropic glutamate receptors (termed mGluR1–mGluR8) (36). These have been classified into three groups based on sequence homology, coupling to second-messenger systems, and pharmacologic sensitivities. Group I receptors are coupled to phosphoinositide (PI) hydrolysis that leads to Ca2+ mobilization from intracellular stores, whereas group II and III receptors are negatively coupled to adenylyl cyclase (AC) activity. Although the consequences of mGluR activation vary depending on receptor type, neuronal type, or brain region, some general principles regarding the effects of mGluR activation in relation to seizures have emerged (37). Postsynaptic group I mGluR activation in general causes an increase in the intrinsic excitability of principal neurons (particularly in hippocampal CA1 and CA3 subfields), mainly by downmodulation of voltage-gated potassium channels (58), and therefore, activation of PI-coupled mGluRs is likely to promote seizure activity. Conversely, presynaptic group II and III receptor activation tends to depress excitatory synaptic transmission by inhibiting glutamate release (59), and therefore, activation of AC-coupled mGluRs is likely to inhibit seizure activity. Given these potential roles for mGluRs in the generation or inhibition of seizure activity, available evidence suggests that the developmental regulation of mGluR function also may contribute to the increased seizure susceptibility of the immature brain. mGluR agonist-stimulated PI turnover has been shown to be relatively robust in slices of immature rat brain, increasing from age P1 to P7–10 before gradually decreasing to adult levels at around P24 (60). This appears to contrast with the activity of mGluRs negatively coupled to AC, as cyclic AMP accumulation induced by the AC activator forskolin was shown to be inhibited by the nonspecific mGluR agonist 1S,3R(ACPD) in adult but not in neonatal (P1–15) rat hippocampus (61,62). Interestingly, mGluRs negatively coupled to AC are expressed in early postnatal development, but nonspecific mGluR activation in the neonatal hippocampus increases basal cyclic AMP levels (63). Thus, regardless of mGluR gene expression, the developmental pattern of mGluR function may favor a hyperexcitable state as the activity of postsynaptic mGluRs that promote increased intrinsic neuronal excitability can predominate over mGluRs that presynaptically regulate neurotransmitter release. The expression of glutamate transporters also is developmentally regulated and may play a role in the enhanced excitability of the immature brain. In animal models, decreased expression of glutamate transporters can lead to seizures or lower seizure thresholds (64). For example, knockout mice lacking the glutamate transporter GLT-1 display lethal seizures that are related to elevated extracellular glutamate levels (65). Additionally, mice deficient in the glutamate transporter GLAST do not exhibit spontaneous seizures, but show shorter onset latency and more severe stages of pentylenetetrazol-induced seizures compared with wild-type mice (66). Notably, the expression of both of these astrocytic glutamate transporters gradually increases during postnatal development in the rat (67). Thus, a relatively lower activity of certain glutamate transporters during development could contribute to enhanced seizure susceptibility in the immature brain. However, the developmental profile of glutamate-transporter expression and regulation differs across transporter subtypes and brain regions (67,68), and the specific contributions of each subtype during development are yet to be fully elucidated. γ-Aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the brain, and the expression and function of GABA receptors also are developmentally regulated. GABAA receptors, which mediate postsynaptic responses to GABA in central neurons, are expressed at embryonic stages (69). However, in the first postnatal week, activation of GABAA receptors causes membrane depolarization rather than the hyperpolarization typical of mature GABA-ergic synapses (see Fig. 1) (28,70). This difference is not due to receptor composition, but rather results from maturational changes in the transmembrane chloride ion gradient, as this largely governs the equilibrium potential for GABAA channels (71). Inhibitory (hyperpolarizing) GABAA receptor–mediated potentials gradually appear over the first 3 postnatal weeks (70)(Fig. 1) and are temporally correlated with the induction of expression of the neuronal K+/Cl– cotransporter KCC2, which extrudes Cl– from cells (72). Thus, although functional GABA receptors are present very early in development, the delayed onset of functional GABA-ergic inhibition may contribute to the enhanced excitability of the immature brain. The molecular composition of GABAA receptors also is developmentally regulated with associated changes in functional properties (73,74). For example, the expression of GABAA receptor α1 subunits is low at birth and gradually increases with maturation (69,75–77). This maturational change is associated with a gradual shift toward the more rapid kinetics and increased sensitivity to certain benzodiazepines [e.g., diazepam (DZP)] that are characteristic of most GABAA receptors in the adult brain (76,78,79). Such a developmental change in the pharmacologic properties of GABAA receptors suggests that the neonatal brain may respond differently from the adult brain to AEDs that act by enhancing GABAA-receptor function. Unlike GABAA receptors, the G protein–coupled GABAB receptors are activated both pre- and postsynaptically, with opposite effects on synaptic transmission (80). Postsynaptic GABAB receptors mediate relatively slowly activating and long-lasting membrane hyperpolarization through the activation of a K+ conductance, whereas the activation of presynaptic GABAB receptors decreases neurotransmitter release through the inhibition of Ca2+ channels (80). In animal models of epilepsy, GABAB receptors can have varied roles depending on the particular model and brain region involved. For example, infusion of a GABAB-receptor antagonist into the substantia nigra decreased thresholds for flurothyl-induced seizures in immature rats (81), whereas GABAB-receptor antagonists abolish seizures in several animal models of absence epilepsy (82,83). GABAB-receptor binding in rat brain increases during postnatal development and peaks in a regionally specific manner during the first 3 weeks, before declining to adult levels (84). In neocortex and thalamus, binding gradually increases to an overshoot at P14 before decreasing to mature levels, whereas peak binding occurs within the first postnatal week in hippocampus (84). Notably, in hippocampus, the presynaptic effects of GABAB-receptor activation are observable earlier in development than are the postsynaptic effects (80). If the activation of presynaptic GABAB receptors depresses GABA release, then the earlier appearance of presynaptic GABAB-receptor function would be expected to promote synaptic excitability. Although a clear role or age dependence of GABAB receptors in the generation or suppression of limbic seizures has not been established (85), GABAB-receptor activation in the thalamus is clearly required for the generation of absence seizures in most animal models, including those that mimic the age dependence of clinical absence epilepsy (83). Thus, it has been suggested that the age dependence of absence seizures may derive from a mismatch in the rates of maturation of GABAB-mediated and glutamate receptor–mediated transmission in thalamocortical circuits such that the maturation of NMDA-receptor function lags behind that of GABAB receptors before adulthood (83). The developmental regulation of certain neuromodulatory peptides also may influence the excitability of the immature brain. The excitatory neuropeptide corticotropin-releasing hormone (CRH) is the most potent epileptogenic peptide (86) and may play a critical role in the "triggering" of seizures (i.e., by fever or hypoxia) in the immature brain (87). In immature rat amygdala, the expression of CRH receptors reaches twice that of the adult level in the second postnatal week, a developmental stage during which the immature brain is most susceptible to CRH-induced seizures (88,89). Other neuromodulatory peptides, such as neuropeptide Y and somatostatin, can modulate seizures (86), and their function and expression are developmentally regulated (90,91). However, specific roles for these neuropeptides in seizures in the immature brain have not been investigated extensively. The expression of epileptiform activity requires synaptic communication among neurons, and therefore, the discussion heretofore has focused on the role of synaptic mechanisms in seizures and epileptogenesis. However, the intrinsic excitability of postsynaptic and axonal membranes is largely determined by the activity of voltage-gated ion channels, and the developmental regulation of voltage-gated channels and ionic homeostasis may contribute to the increased seizure susceptibility of the immature brain. The developmental patterns of expression and function of voltage-gated ion channels vary across cell type and brain region. In general, these patterns promote increased intrinsic membrane excitability during embryogenesis and early postnatal development, as spontaneous electrical activity is necessary for cell differentiation, migration, and synaptogenesis (92). Action potentials in immature neurons are of significantly longer duration compared with their adult counterparts, largely due to slower activation rates of delayed rectifier K+ channels that repolarize the action potential (92,93). During an action potential, the amount of Ca2+ influx through voltage-gated Ca2+ channels is determined largely by action-potential duration, and therefore, the longer action potentials in immature neurons play a key role in activity-driven Ca2+-dependent developmental events. For example, premature shortening of the action potential by overexpression of a delayed-rectifier K+ channel (Kv1.1) in embryonic Xenopus spinal neurons caused a decrease in the number of neurons achieving terminal differentiation (94). Notably, knockout mice lacking Kv1.1 exhibit spontaneous seizures beginning in the third postnatal week (95). Clearly, the increased intrinsic membrane excitability in immature neurons is critical for normal development, but at the apparent expense of enhancing network excitability while development is ongoing. The developmental regulation of voltage-gated channels in axons and at presynaptic terminals may be particularly critical in the generation of seizure activity through their influence on neurotransmitter release. In epileptic DBA/2J mice, a strain genetically prone to audiogenic seizures, binding of a radiolabeled N-type Ca2+-channel toxin (ω-conotoxin GVIA) indicated that the expression of these presynaptic Ca2+ channels was abnormally increased between postnatal days 2–8 in parallel with seizure susceptibility (96). In contrast, nonepileptic mice showed no change in ω-GVIA binding during the same period, but exhibited a rapid increase in binding between postnatal days 11 and 14 (97). Notably, although these mice are not epileptic, this period of N-channel proliferation coincides with the period of heightened susceptibility in rodents to seizures induced by hypoxia or hyperthermia, as discussed later. Thus, the developmental change in presynaptic Ca2+ channels that govern neurotransmitter release may be a critical component in determining the early postnatal susceptibility to seizure. Mechanisms of ion homeostasis also are changing during development. As mentioned earlier, the neuronal expression of chloride transporters has a postnatal onset, and in their absence, the transmembrane Cl– gradient is such that Cl–-mediated currents are depolarizing. The major neuronal Na+/K+ adenosine triphosphatase (ATPase) also is less abundant in the immature brain (98), and thus, moderate increases in neuronal activity could regeneratively cause extracellular K+ to increase to epileptogenic levels. Intracellular Ca2+ homeostasis also may be developmentally regulated. For example, the calcium-binding protein calbindin D28K is not expressed in immature hippocampal dentate granule cells, but is expressed by these same cells as they mature (99). A number of experimental seizure models have at least in part recapitulated components of the age-specific seizure syndromes (100–102). Symptomatic early-onset seizures can be modeled in the developing brain by exposure to chemoconvulsants such as kainate, pilocarpine, flurothyl, and tetanus toxin, or by repeated electrical stimulation (kindling). Seizure thresholds to chemoconvulsant and kindling stimuli are much lower in immature animals (100,103). Additionally, certain pathophysiologic conditions such as hypoxia or hyperthermia may precipitate seizures in the immature animal, while having no epileptogenic effect in the adult (104,105). As mentioned earlier, CRH can trigger spontaneous seizures in immature animals, but not in adults (87). In general, these models exhibit age dependence not only in their efficacy for inducing seizures, but also in their long-term effects on brain function. Unlike that in the adult, the immature brain appears to be relatively resistant to seizure-induced neuronal injury (1,102). Kainate injections induce status epilepticus in immature animals with no delayed neuronal loss despite ≥2 h of continuous seizures (106–109). More severe epileptogenic conditions have been found to both induce seizures and cause neuronal injury in the immature brain (110), and pilocarpine status-induced neuronal death in the immature brain has been reported (111). However, the extent of death is significantly less than that seen after status in the adult brain (112). These data suggest that although the resistance of the immature brain to chemoconvulsant-induced neuronal death is not absolute, it is much greater relative to the adult. Despite the lack of injury in the immature brain in these experimental models, other evidence suggests that neonatal seizures may adversely alter the function of neurons and neuronal to seizure thresholds or to promote epileptogenesis. In rat at age hypoxia spontaneous seizures and results in decreased thresholds to chemoconvulsant-induced seizures and increased excitability in hippocampal slices despite no apparent neuronal injury convulsions in the P10 rat do not cause acute cell yet in long-lasting changes in inhibitory synaptic transmission and decreased seizure and increased hippocampal excitability tetanus toxin injections in P10 rat in the development of acute seizures, which lead to spontaneous recurrent seizures later in life and in the dentate seizures induced by repeated or beginning in the second postnatal week in long-term increases in seizure susceptibility with in hippocampal Notably, animals that repeated flurothyl-induced seizures also showed but impairment in and in adulthood The cellular and molecular events that mediate these and functional changes are but these data the that seizures can induce adverse functional changes in the immature brain that may not appear as of the epileptic mature brain have revealed the contributions of cellular and molecular factors to seizure expression and epilepsy. Given this can at how the functional properties of many of these factors change with maturation, and the potential consequences for seizure susceptibility at different developmental Although developmental changes can alter seizure seizures also may alter the normal pattern of brain development. To age-specific for the treatment of neonatal and childhood seizures, it will be necessary to identify developmentally regulated factors that are critical for acute and long-term seizure and to understand how brain maturation may be altered by The developmental profile of KA receptor function in Fig. was based on receptor binding In has shown a more varied maturational pattern of KA receptor subunit gene expression, with gene expression at birth in thalamus, and hippocampal CA1 et evidence that low KA receptors are activated at thalamocortical synapses during early postnatal development and and therefore, may have a role in synaptic signaling and plasticity in the immature brain. on possible roles for these receptors in determining seizure susceptibility further This was supported by from the on from the of and and the from the of and

Introduction. Posttraumatic epilepsy: treatable epileptogenesis.

ABSTRACTEpilepsy is a neurological disease that is caused by abnormal hypersynchronous activities of neuronal ensembles leading to recurrent and spontaneous seizures in human patients. Enhanced neuronal excitability and a high level of synchrony between neurons seem to trigger these spontaneous seizures. The molecular mechanisms, however, regarding the development of neuronal hyperexcitability and maintenance of epilepsy are still poorly understood. Here, we show that pumilio RNA-binding family member 2 (Pumilio2; Pum2) plays a role in the regulation of excitability in hippocampal neurons of weaned and 5-month-old male mice. Almost complete deficiency of Pum2 in adult Pum2 gene-trap mice (Pum2 GT) causes misregulation of genes involved in neuronal excitability control. Interestingly, this finding is accompanied by the development of spontaneous epileptic seizures in Pum2 GT mice. Furthermore, we detect an age-dependent increase in Scn1a (Nav1.1) and Scn8a (Nav1.6) mRNA levels together with a decrease in Scn2a (Nav1.2) transcript levels in weaned Pum2 GT that is absent in older mice. Moreover, field recordings of CA1 pyramidal neurons show a tendency towards a reduced paired-pulse inhibition after stimulation of the Schaffer-collateral-commissural pathway in Pum2 GT mice, indicating a predisposition to the development of spontaneous seizures at later stages. With the onset of spontaneous seizures at the age of 5 months, we detect increased protein levels of Nav1.1 and Nav1.2 as well as decreased protein levels of Nav1.6 in those mice. In addition, GABA receptor subunit alpha-2 (Gabra2) mRNA levels are increased in weaned and adult mice. Furthermore, we observe an enhanced GABRA2 protein level in the dendritic field of the CA1 subregion in the Pum2 GT hippocampus. We conclude that altered expression levels of known epileptic risk factors such as Nav1.1, Nav1.2, Nav1.6 and GABRA2 result in enhanced seizure susceptibility and manifestation of epilepsy in the hippocampus. Thus, our results argue for a role of Pum2 in epileptogenesis and the maintenance of epilepsy.

Stargazin is membrane bound protein involved in trafficking, synapse anchoring and biophysical modulation of AMPA receptors. A quantitative trait locus in chromosome 7 containing the stargazin gene has been identified as controlling the frequency and duration of absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Furthermore, mutations in this gene result in the Stargazer mouse that displays an absence epilepsy phenotype. GAERS stargazin mRNA expression is increased 1.8 fold in the somatosensory cortex and by 1.3 fold in the thalamus. The changes were present before and after the onset of absence seizures indicating that increases are not a secondary consequence of the seizures. Stargazin protein expression was also significantly increased in the somatosensory cortex after the onset of spontaneous seizures. The results are of significant importance beyond the GAERS model, as they are the first to show that an increase in stargazin expression may be pro-epileptic.

Epilepsies affect about 4% of the population and are frequently characterized by a prolonged “silent” period before the onset of spontaneous seizures. Most current animal models of epilepsy either involve acute seizure induction or kindling protocols that induce repetitive seizures. We have developed a rat model of epilepsy that is characterized by a slowly progressing series of behavioral abnormalities prior to the onset of behavioral seizures. In the current study, we further describe an accompanying progression of cytoarchitectural changes in the hippocampal formation. Groups of male and female SD rats received serial injections of a low dose of domoic acid (0.020 mg/kg) (or vehicle) throughout the second week of life. Postmortem hippocampal tissue was obtained on postnatal days 29, 64, and 90 and processed for glial fibrillary acidic protein (GFAP), NeuN, and calbindin expression. The data revealed no significant changes on postnatal day (PND) 29 but a significant increase in hilar NeuN-positive cells in some regions on PND 64 and 90 that were identified as ectopic granule cells. Further, an increase in GFAP positive cell counts and evidence of reactive astrogliosis was found on PND 90 but not at earlier time points. We conclude that changes in cellular expression, possibly due to on-going non-convulsive seizures, develop slowly in this model and may contribute to progressive brain dysfunction that culminates in a seizure-prone phenotype.

We determined the role of the neurosteroid-sensitive δ subunit-containing γ-aminobutyric acid A receptors (δ-GABARs) in epileptogenesis. Status epilepticus (SE) was induced via lithium pilocarpine in adult rats, and seizures were assessed by continuous video-electroencephalography (EEG) monitoring. Finasteride was administered to inhibit neurosteroid synthesis. The total and surface protein expression of hippocampal δ, α4, and γ2 GABAR subunits was studied using biotinylation assays and Western blotting. Neurosteroid potentiation of the tonic currents of dentate granule cells (DGCs) was measured by whole-cell patch-clamp technique. Finally, the effects of inhibiting N-methyl-d-aspartate receptors (NMDARs) during SE on the long-term plasticity of δ-GABARs, neurosteroid-induced modulation of tonic current, and epileptogenesis were studied. The inhibition of neurosteroid synthesis 4 days after SE triggered acute seizures and accelerated the onset of chronic recurrent spontaneous seizures (epilepsy). The down-regulation of neurosteroid-sensitive δ-GABARs occurred prior to the onset of epilepsy, whereas an increased expression of the γ2-GABAR subunits occurred after seizure onset. MK801 blockade of NMDARs during SE preserved the expression of neurosteroid-sensitive δ-GABARs. NMDAR blockade during SE also prevented the onset of spontaneous seizures. Changes in neurosteroid-sensitive δ-GABAR expression correlated temporally with epileptogenesis. These findings raise the possibility that δ-GABAR plasticity may play a role in epileptogenesis.