Abstract
Enduring Memory Impairments Provoked by Developmental Febrile Seizures Are Mediated by Functional and Structural Effects of Neuronal Restrictive Silencing Factor
Mediated by Functional and Structural Effects of Neuronal Restrictive Silencing Factor Patterson KP, Barry JM, Curran MM, Singh-Taylor A, Brennan G, Rismanchi N, Page M, Noam Y, Holmes GL, Baram TZ. J Neurosci 2017;37:3799–3812.
In a subset of children experiencing prolonged febrile seizures (FSs), the most common type of childhood seizures, cognitive outcomes are compromised. However, the underlying mechanisms are unknown. Here we identified significant, enduring spatial memory problems in male rats following experimental prolonged FS (febrile status epilepticus; eFSE). Remarkably, these deficits were abolished by transient, post hoc interference with the chromatin binding of the transcriptional repressor neuron restrictive silencing factor (NRSF or REST). This transcriptional regulator is known to contribute to neuronal differentiation during development and to programmed gene expression in mature neurons. The mechanisms of the eFSE-provoked memory problems involved complex disruption of memory related hippocampal oscillations recorded from CA1, likely resulting in part from impairments of dendritic filtering of cortical inputs as well as abnormal synaptic function. Accordingly, eFSE provoked region-specific dendritic loss in the hippocampus, and aberrant generation of excitatory synapses in dentate gyrus granule cells. Blocking NRSF transiently after eFSE prevented granule cell dysmaturation, restored a functional balance of gamma-band network oscillations, and allowed treated eFSE rats to encode and retrieve spatial memories. Together, these studies provide novel insights into developing networks that underlie memory, the mechanisms by which early-life seizures influence them, and the means to abrogate the ensuing cognitive problems.
Commentary
As the King (of rock'n roll) once said: “everybody's got the fever” during childhood. Fever is one of the most common presenting signs of illness in pediatric practice, and it is an aspect of 19% to 30% of all encounters (1). However, in only a small proportion of children is the fever associated with seizures: In the United States and Western Europe, 2 to 5 percent of children will have experienced at least one febrile seizure by the age of 5 years. This proportion is higher in Japan (6–9%) and even higher in some geographical locations, such as India or Guam (2). As much as one would like to say that the quality of febrile seizures in children may differ, it is the duration of febrile seizures that eventually makes the quality leap in terms of outcome: The population of children with febrile seizures can be separated into two distinct subgroups. One subpopulation is responsible for about 82% of all febrile seizures, and their duration is a mean of 3.8 minutes. The second subpopulation accounts for about 18% of the total, and the mean duration of febrile seizures is approximately 40 minutes (3), thereby fulfilling the definition of febrile status epilepticus (i.e., duration >30 min). Febrile status epilepticus may result in hippocampal sclerosis (4) and in the development of temporal lobe epilepsy, which may further fuel hippocampal sclerosis. Hippocampal damage will eventually be reflected in impaired cognitive abilities (such as reference 5). The mechanisms of febrile status epilepticus involvement are unclear, but the gravity of clinical importance is immense (6). Hence, an experimental approach that could highlight mechanisms of the axis febrile status epilepticus, hippocampal damage, and cognitive impairment is timely, extremely important, and sought after.
The current study is building on the vast expertise of the authors with the model of febrile seizures in immature rats as well as some essential mechanisms found in this model. The authors are more focused on the cognitive impairments and their potential mechanisms that are affiliated with febrile status epilepticus rather than on febrile seizures per se. This is quite understandable. Febrile status epilepticus is a long, but transient, event. On the other hand, cognitive impairments resulting from hippocampal damage related to the original developmental insult (here febrile status epilepticus) or ensuing epilepsy later in life are permanent and significantly contributing to poor quality of life (see reference 7 as a refresher).
The authors hypothesized that the insult by febrile status epilepticus induces neuron restrictive silencing factor (NRSF), a transcription factor (8), which in excess may contribute to cognitive decline. NRSF increases are observed in the hippocampus after epilepsy-inducing insults. Accordingly, inhibition of NRSF chromatin binding after status epilepticus may attenuate the development of epilepsy. All of these actions of NRSF may be mediated via its regulation of gene expression programs during neuronal maturations with HCN1 chromatin playing a prominent role among hundreds of regulated target genes (9). As febrile status epilepticus occurs (or is induced) during the hippocampal developmental period with still ongoing differentiation of dentate gyrus granule cells, these may be very susceptible to fluctuations of NRSF levels. Hence, the focus of the study was on the hippocampal granular cell network.
The authors used a simple yet powerful approach based on their model of neonatal hyperthermia in rats (10). Hyperthermia (core temperature between 39.5 °C and 41.0°C) was induced and maintained in half of P10-P11 rat pups from the onset of seizures for 60 minutes. The other half of the pups served as nonhyperthermic controls. Then half of each group received an NRSE (nucleotide recognition element for NRSF to provide a decoy target for excess NRSF) sequenced oligonucleotide icv and the other half received scrambled sequence of the NRS oligonucleotide. This two-factor design was able to control for main effects, hyperthermic seizure and NRSF function, by flooding with false targets (NRSE oligonucleotides). Animals from these four groups were kept for 3 to 5 months (until adulthood) and then used in multiple experiments. Initially, cognition and spatial orientation (active avoidance [11]) were assessed together with local field potential activity of granule cells in the hippocampal dentate gyrus. After febrile status epilepticus, animals had problems with spatial orientation in the active avoidance task consisting of avoiding a shock zone based on spatial cues. Interestingly, their rate of learning, i.e., slope of learning curves, was similar to other groups. Memory retention was also impaired since at retesting 1 month later they were unable to remember the location of the shock zone, unlike the other groups. If NRSF function was blocked by NRSE flood, cognitive deficits assessed months after febrile status epilepticus were attenuated. Concurrent recording of local field potentials revealed occurrence of high-amplitude fast gamma oscillations consistent with failure of filtering of entorhino-cortical to CA1 place cell inputs (temporo-ammonic pathway). On the other hand, slow gamma oscillations were suppressed, reflecting impairment of dentate gyrus gating on CA3 to CA1 inputs. Inhibition of NRSF function was capable of rectifying these deficits. There was also a long-term increase in NRSF levels and in its binding to chromatin after febrile status epilepticus, which persisted for 2 months. Specifically, binding of NRSF to HCN1 chromatin was profoundly increased. Impaired HCN1 currents are hallmarks of many seizure types (9, 12), and HCN1 is reduced after early life seizures, including genetic epilepsies (13). HCN1 seems to play a role in cognitive deficits as well (14). Examination of the effect of NRSF enhancement by febrile status epilepticus on morphologic development of granule cells (Scholl analysis in Golgi impregnation) uncovered enrichment of dendritic branching of granule cells that was sensitive to NRSE oligonucleotide inhibition. After febrile status epilepticus, this enhanced branching may potentially give rise to aberrant synapses on expanded dendrites of granule cells. In a complementary experiment, neonatal (P7) mice with YFP tag of granule cells (Thy1 promoter) were used to prepare organotypic hippocampal slices to determine effects of kainic acid in vitro “seizures” and the role of NRSF in these seizures. These in vitro seizures led to retention of hilar basal dendrites of granule cells, normally lost with maturation, which form excitatory synapses on neighboring granule cells and thus, provide excessive dentate gyrus drive.
The study demonstrates that developmental febrile status epilepticus may lead to abnormal maturation of granule cells and may distort development of entire hippocampal network with significant contribution to cognitive impairments. The largest impact of this study is that post hoc (after the febrile status epilepticus) intervention leading to suppression of NRSF binding to chromatin may significantly attenuate these mechanisms and outcomes. NRSF appears to be a very potent mediator of seizure-induced deficits in the dentate gyrus network, which is crucial for gating information flow to the hippocampus proper. Defects in this gating may significantly contribute not only to behavioral/cognitive deficits but also to epileptogenesis (15).
Indeed, questions persist: A previous study of the core group indicated that febrile status epilepticus of a similar duration (64 minutes vs 60 minutes here) resulted in significant epileptogenesis (45% of animals [16]). Hence the question: How would it be possible to separate cognitive effects of the primary insult (here febrile status epilepticus) from the contribution of spontaneous seizures resulting from that insult (in at least 45% of the subjects). This potential confounding effect of spontaneous seizures on cognitive parameters needs to be addressed. The question can be also reversed. If NRSF function is blocked by decoy target molecule, is there still epileptogenesis; in other words, is it the epileptogenesis providing the lion's share of cognitive defects? Finally, the comparison of the effects of a brief febrile seizure (about 4 minutes) versus long (60 minutes as used here) would offer additional support for differential approaches (if differences in cognition and mechanism were found) to brief febrile seizures and febrile status epilepticus.