Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (I(H)). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.

SUMMARY Adult hippocampal neurogenesis is believed to maintain a range of cognitive functions, many of which decline with age. We recently reported that radial neural stem cells (rNSCs) in the hippocampus undergo activation-dependent conversion into astrocytes, a mechanism that over time contributes to a reduction in the rNSC population. Here, we injected low and high levels of kainic acid (KA) in the dentate gyrus to assess whether neuronal hyperexcitation, a hallmark of epileptic disorders, could accelerate this conversion. At low levels of KA, generating epileptiform activity without seizures, we indeed found increased rNSC activation and conversion into astrocytes. At high levels, generating sustained epileptic seizures, however, we find that rNSCs divide symmetrically and that both mother and daughter cells convert into reactive astrocytes. Our results demonstrate that a threshold response for neuronal hyperexcitation provokes a dramatic shift in rNSCs function, which impairs adult hippocampal neurogenesis in the long term.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Changes in the expression of ion channels, contributing to altered neuronal excitability, are emerging as possible mechanisms in the development of certain human epilepsies. In previous immature rodent studies of experimental prolonged febrile seizures, isoform-specific changes in the expression of hyperpolarization-activated cyclic nucleotide-gated cation channels (HCNs) correlated with long-lasting hippocampal hyperexcitability and enhanced seizure susceptibility. Prolonged early-life seizures commonly precede human temporal lobe epilepsy (TLE), suggesting that transcriptional dysregulation of HCNs might contribute to the epileptogenic process. Therefore, we determined whether HCN isoform expression was modified in hippocampi of individuals with TLE. HCN1 and HCN2 expression were measured using in situ hybridization and immunocytochemistry in hippocampi from three groups: TLE with hippocampal sclerosis (HS; n = 17), epileptic hippocampi without HS, or non-HS (NHS; n = 10), and autopsy material (n = 10). The results obtained in chronic human epilepsy were validated by examining hippocampi from the pilocarpine model of chronic TLE. In autopsy and most NHS hippocampi, HCN1 mRNA expression was substantial in pyramidal cell layers and lower in dentate gyrus granule cells (GCs). In contrast, HCN1 mRNA expression over the GC layer and in individual GCs from epileptic hippocampus was markedly increased once GC neuronal density was reduced by >50%. HCN1 mRNA changes were accompanied by enhanced immunoreactivity in the GC dendritic fields and more modest changes in HCN2 mRNA expression. Furthermore, similar robust and isoform-selective augmentation of HCN1 mRNA expression was evident also in the pilocarpine animal model of TLE. These findings indicate that the expression of HCN isoforms is dynamically regulated in human as well as in experimental hippocampal epilepsy. After experimental febrile seizures (i.e., early in the epileptogenic process), the preserved and augmented inhibition onto principal cells may lead to reduced HCN1 expression. In contrast, in chronic epileptic HS hippocampus studied here, the profound loss of interneuronal and principal cell populations and consequent reduced inhibition, coupled with increased dendritic excitation of surviving GCs, might provoke a "compensatory" enhancement of HCN1 mRNA and protein expression.

Seizures induced by fever (febrile seizures) are the most common type of pathological brain activity in infants and children. These febrile seizures and their potential contribution to the mechanisms of limbic (temporal lobe) epilepsy have been a topic of major clinical and scientific interest. Key questions include the mechanisms by which fever generates seizures, the effects of long febrile seizures on neuronal function and the potential contribution of these seizures to epilepsy. This review builds on recent advances derived from animal models and summarizes our current knowledge of the mechanisms underlying febrile seizures and of changes in neuronal gene expression and function that facilitate the enduring effects of prolonged febrile seizures on neuronal and network excitability. The review also discusses the relevance of these findings to the general mechanisms of epileptogenesis during development and points out gaps in our knowledge, including the relationship of animal models to human febrile seizures and epilepsy.

Cognitive impairments are prominent sequelae of prolonged continuous seizures (status epilepticus; SE) in humans and animal models. While often associated with dendritic injury, the underlying mechanisms remain elusive. The mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated following SE. This pathway modulates learning and memory and is associated with regulation of neuronal, dendritic, and glial properties. Thus, in the present study we tested the hypothesis that SE-induced mTORC1 hyperactivation is a candidate mechanism underlying cognitive deficits and dendritic pathology seen following SE. We examined the effects of rapamycin, an mTORC1 inhibitor, on the early hippocampal-dependent spatial learning and memory deficits associated with an episode of pilocarpine-induced SE. Rapamycin-treated SE rats performed significantly better than the vehicle-treated rats in two spatial memory tasks, the Morris water maze and the novel object recognition test. At the molecular level, we found that the SE-induced increase in mTORC1 signaling was localized in neurons and microglia. Rapamycin decreased the SE-induced mTOR activation and attenuated microgliosis which was mostly localized within the CA1 area. These findings paralleled a reversal of the SE-induced decreases in dendritic Map2 and ion channels levels as well as improved dendritic branching and spine density in area CA1 following rapamycin treatment. Taken together, these findings suggest that mTORC1 hyperactivity contributes to early hippocampal-dependent spatial learning and memory deficits and dendritic dysregulation associated with SE.

Hyperpolarization-activated cation currents (I(h)) are found in several brain regions including thalamus and hippocampus. Important functions of these currents in promoting synchronized network activity and in determining neuronal membrane properties have been progressively recognized, but the molecular underpinnings of these currents are only emerging. I(h) currents are generated by hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs). These channel proteins are encoded by at least four HCN genes, that govern the kinetic and functional properties of the resulting channels. Because of the potential impact of I(h)-mediated coordinated neuronal activity on the maturation of the functional hippocampal network, this study focused on determining the expression of the four members of the HCN gene family throughout postnatal hippocampal development at both the regional and single cell level.The results of these experiments demonstrated that HCNs 1, 2 and 4 are differentially expressed in interneuronal and principal cell populations of the rat hippocampal formation. Expression profiles of each HCN isoform evolve during postnatal development, and patterns observed during early postnatal ages differ significantly from those in mature hippocampus. The onset of HCN expression in interneurons of the hippocampus proper precedes that in the dentate gyrus, suggesting that HCN-mediated pacing activity may be generated in hippocampal interneurons prior to those in the hilus. Taken together, these findings indicate an age-dependent spatiotemporal evolution of specific HCN expression in distinct hippocampal cell populations, and suggest that these channels serve differing and evolving functions in the maturation of coordinated hippocampal activity.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate hyperpolarizationactivated currents (I h ). In hippocampus, these currents contribute greatly to intrinsic cellular properties and synchronized neuronal activity. The kinetic and gating properties of HCN-mediated currents are largely determined by the type of subunits-for example, HCN1 and HCN2-that assemble to form homomeric channels. Recently, functional heteromeric HCN channels have been described in vitro, further enlarging the potential I h repertoire of individual neurons. Because these heteromeric HCN channels may promote hippocampal hyperexcitability and the development of epilepsy, understanding the mechanisms governing their formation is of major clinical relevance. Here, we find that developmental seizures promote co-assembly of hippocampal HCN1/HCN2 heteromeric channels, in a duration-dependent manner. Long-lasting heteromerization was found selectively after seizures that provoked persistent hippocampal hyperexcitability. The mechanism for this enhanced heteromerization may involve increased relative abundance of HCN2-type subunits relative to the HCN1 isoform at both mRNA and protein levels. These data suggest that heteromeric HCN channels may provide molecular targets for intervention in the epileptogenic process.