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Developmental and epileptic encephalopathies (DEE) are rare but devastating and largely intractable childhood epilepsies. Genetic variants in ARHGEF9 , encoding a scaffolding protein important for the organization of the postsynaptic density of inhibitory synapses, are associated with DEE accompanied by complex neurological phenotypes. In a mouse model carrying a patient-derived ARHGEF9 variant associated with severe disease, we observed aggregation of postsynaptic proteins and loss of functional inhibitory synapses at the axon initial segment (AIS), altered axo-axonic synaptic inhibition, disrupted action potential generation, and complex seizure phenotypes consistent with clinical observations. These results illustrate diverse roles of ARHGEF9 that converge on regulation of the structure and function of the AIS, thus revealing a pathological mechanism for ARHGEF9 -associated DEE. This unique example of a neuropathological condition associated with multiple AIS dysfunctions may inform strategies for treating neurodevelopmental diseases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wild-type replacement. We evaluated such a knockdown-replace strategy in a mouse model of DNM1 disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons-which resulted in growth delay and lethal seizures evident by postnatal week three-and delivered to newborn pups an AAV9-based vector encoding a ubiquitously expressed, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 cDNA. Pups receiving RNAi or cDNA alone fared no better than untreated pups, whereas the vast majority of mutants receiving modest doses survived with almost full growth recovery. Synaptic recordings of cortical neurons derived from treated pups revealed that significant alterations in transmission from inhibitory to excitatory neurons were rectified by bivalent vector application. To examine the mutant transcriptome and impact of treatment, we used RNA sequencing and functional annotation clustering. Mutants displayed abnormal expression of more than 1,000 genes in highly significant and relevant functional clusters, clusters that were abrogated by treatment. Together these results suggest knockdown-replace as a potentially effective strategy for treating DNM1 and related genetic neurodevelopmental disease., Competing Interests: Declaration of interests A patent application was submitted (Appl. No. 63/639,576: Products and methods to inhibit expression of dynamin-1 variants and replace dynamin-1). Inventors: S.Q.H., N.T., and W.N.F., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

More than twenty recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (K Na ) channel gene KCNT1 in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a Kcnt1 GOF variant (Y777H) on K Na currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant KCNT1 GOF variants in DEEs. Although the Kcnt1 -Y777H variant had no effects on glutamatergic or VIP neuron function, it increased subthreshold K Na currents in both SST and PV neurons but with opposite effects on neuronal output; SST neurons became hypoexcitable with a higher rheobase current and lower action potential (AP) firing frequency, whereas PV neurons became hyperexcitable with a lower rheobase current and higher AP firing frequency. Further neurophysiological and computational modeling experiments showed that the differential effects of the Y777H variant on SST and PV neurons are not likely due to inherent differences in these neuron types, but to an increased persistent sodium current in PV, but not SST, neurons. The Y777H variant also increased excitatory input onto, and chemical and electrical synaptic connectivity between, SST neurons. Together, these data suggest differential pathogenic mechanisms, both direct and compensatory, contribute to disease phenotypes, and provide a salient example of how a pathogenic ion channel variant can cause opposite functional effects in closely related neuron subtypes due to interactions with other ionic conductances., Competing Interests: Conflict of interest statement: The authors declare no competing financial interests.

Heterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the potential groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated global developmental delay, impaired ultrasonic vocalizations, cognitive dysfunction and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the subiculum-a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlights the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPU-mediated disease mechanisms and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests. D.B.G. is a founder of Actio Biosciences, a founder of and holds equity in Praxis, serves as a consultant to AstraZeneca, and has received research support from Janssen, Gilead, Biogen, AstraZeneca and UCB. R.S.D serves as a consultant to AstraZeneca. S.C. serves a consultant for Q-State Biosciences, Inc., (Copyright: © 2023 Dugger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

De novo mutations in GNB1 , encoding the G β 1 subunit of G proteins, cause a neurodevelopmental disorder with global developmental delay and epilepsy, GNB1 encephalopathy. Here, we show that mice carrying a pathogenic mutation, K78R, recapitulate aspects of the disorder, including developmental delay and generalized seizures. Cultured mutant cortical neurons also display aberrant bursting activity on multi-electrode arrays. Strikingly, the antiepileptic drug ethosuximide (ETX) restores normal neuronal network behavior in vitro and suppresses spike-and-wave discharges (SWD) in vivo . ETX is a known blocker of T-type voltage-gated Ca 2+ channels and G protein-coupled potassium (GIRK) channels. Accordingly, we present evidence that K78R results in a gain-of-function (GoF) effect by increasing the activation of GIRK channels in cultured neurons and a heterologous model ( Xenopus oocytes)-an effect we show can be potently inhibited by ETX. This work implicates a GoF mechanism for GIRK channels in epilepsy, identifies a new mechanism of action for ETX in preventing seizures, and establishes this mouse model as a pre-clinical tool for translational research with predicative value for GNB1 encephalopathy., Competing Interests: DG is a founder of Actio Biosciences, a founder of and holds equity in Praxis, serves as a consultant to AstraZeneca, and has received research support from Janssen, Gilead, Biogen, AstraZeneca and UCB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Colombo, Reddy, Petri, Williams, Shalomov, Dhindsa, Gelfman, Krizay, Bera, Yang, Peng, Makinson, Boland, Frankel, Goldstein and Dascal.)

Absence seizures, manifested by spike-wave discharges (SWD) in the electroencephalogram, display synchronous reciprocal excitation between the neocortex and thalamus. Recent studies have revealed that inhibitory neurons in the reticular thalamic (RT) nucleus and excitatory thalamocortical (TC) neurons are two subcortical players in generating SWD. However, the signals that drive SWD-related activity remain elusive. Here, we show that SWD predominately occurs during wakefulness in several mouse models of absence epilepsy. In more focused studies of Gnb1 mutant mice, we found that sensory input regulates SWD. Using in vivo recording, we demonstrate that TC cells are activated prior to the onset of SWD and then inhibited during SWD. On the contrary, RT cells are slightly inhibited prior to SWD, but are strongly activated during SWD. Furthermore, chemogenetic activation of TC cells leads to the enhancement of SWD. Together, our results indicate that sensory input can regulate SWD by activating the thalamocortical pathway., Competing Interests: Authors declare that they have no competing interests., (© 2022 The Author(s).)

While several studies have attributed the development of tumour-associated seizures to an excitatory-inhibitory imbalance, we have yet to resolve the spatiotemporal interplay between different types of neuron in glioma-infiltrated cortex. Herein, we combined methods for single unit analysis of microelectrode array recordings with wide-field optical mapping of Thy1-GCaMP pyramidal cells in an ex vivo acute slice model of diffusely infiltrating glioma. This enabled simultaneous tracking of individual neurons from both excitatory and inhibitory populations throughout seizure-like events. Moreover, our approach allowed for observation of how the crosstalk between these neurons varied spatially, as we recorded across an extended region of glioma-infiltrated cortex. In tumour-bearing slices, we observed marked alterations in single units classified as putative fast-spiking interneurons, including reduced firing, activity concentrated within excitatory bursts and deficits in local inhibition. These results were correlated with increases in overall excitability. Mechanistic perturbation of this system with the mTOR inhibitor AZD8055 revealed increased firing of putative fast-spiking interneurons and restoration of local inhibition, with concomitant decreases in overall excitability. Altogether, our findings suggest that diffusely infiltrating glioma affect the interplay between excitatory and inhibitory neuronal populations in a reversible manner, highlighting a prominent role for functional mechanisms linked to mTOR activation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tuberous sclerosis complex (TSC) is a developmental disorder associated with epilepsy, autism, and cognitive impairment. Despite inactivating mutations in the TSC1 or TSC2 genes and hyperactive mechanistic target of rapamycin (mTOR) signaling, the mechanisms underlying TSC-associated neurological symptoms remain incompletely understood. Here we generate a Tsc1 conditional knockout (CKO) mouse model in which Tsc1 inactivation in late embryonic radial glia causes social and cognitive impairment and spontaneous seizures. Tsc1 depletion occurs in a subset of layer 2/3 cortical pyramidal neurons, leading to development of cytomegalic pyramidal neurons (CPNs) that mimic dysplastic neurons in human TSC, featuring abnormal dendritic and axonal overgrowth, enhanced glutamatergic synaptic transmission, and increased susceptibility to seizure-like activities. We provide evidence that enhanced synaptic excitation in CPNs contributes to cortical hyperexcitability and epileptogenesis. In contrast, astrocytic regulation of synapse formation and synaptic transmission remains unchanged after late embryonic radial glial Tsc1 inactivation, and astrogliosis evolves secondary to seizures., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Developmental epileptic encephalopathies (DEEs) are severe seizure disorders that occur in infants and young children, characterized by developmental delay, cognitive decline, and early mortality. Recent efforts have identified a wide variety of genetic variants that cause DEEs. Among these, variants in the DNM1 gene have emerged as definitive causes of DEEs, including infantile spasms and Lennox-Gastaut syndrome. A mouse model of Dnm1 -associated DEE, known as "Fitful" ( Dnm1 Ftfl ), recapitulates key features of the disease, including spontaneous seizures, early lethality, and neuronal degeneration. Previous work showed that DNM1 is a key regulator of synaptic vesicle (SV) endocytosis and synaptic transmission and suggested that inhibitory neurotransmission may be more reliant on DNM1 function than excitatory transmission. The Dnm1 Ftfl variant is thought to encode a dominant negative DNM1 protein; however, the effects of the Dnm1 Ftfl variant on synaptic transmission are largely unknown. To understand these synaptic effects, we recorded from pairs of cultured mouse cortical neurons and characterized all four major connection types [excitation of excitation (E-E), inhibition of inhibition (I-I), E-I, I-E]. Miniature and spontaneous EPSCs and IPSCs were larger, but less frequent, at all Dnm1 Ftfl synaptic types, and Dnm1 Ftfl neurons had reduced expression of excitatory and inhibitory SV markers. Baseline evoked transmission, however, was reduced only at inhibitory synapses onto excitatory neurons, because of a smaller pool of releasable SVs. In addition to these synaptic alterations, Dnm1 Ftfl neurons degenerated later in development, although their activity levels were reduced, suggesting that Dnm1 Ftfl may impair synaptic transmission and neuronal health through distinct mechanisms., (Copyright © 2021 McCabe et al.)

Paucity of the glucose transporter-1 (Glut1) protein resulting from haploinsufficiency of the SLC2A1 gene arrests cerebral angiogenesis and disrupts brain function to cause Glut1 deficiency syndrome (Glut1 DS). Restoring Glut1 to Glut1 DS model mice prevents disease, but the precise cellular sites of action of the transporter, its temporal requirements, and the mechanisms linking scarcity of the protein to brain cell dysfunction remain poorly understood. Here, we show that Glut1 functions in a cell-autonomous manner in the cerebral microvasculature to affect endothelial tip cells and, thus, brain angiogenesis. Moreover, brain endothelial cell-specific Glut1 depletion not only triggers a severe neuroinflammatory response in the Glut1 DS brain, but also reduces levels of brain-derived neurotrophic factor (BDNF) and causes overt disease. Reduced BDNF correlated with fewer neurons in the Glut1 DS brain. Controlled depletion of the protein demonstrated that brain pathology and disease severity was greatest when Glut1 scarcity was induced neonatally, during brain angiogenesis. Reducing Glut1 at later stages had mild or little effect. Our results suggest that targeting brain endothelial cells during early development is important to ensure proper brain angiogenesis, prevent neuroinflammation, maintain BDNF levels, and preserve neuron numbers. This requirement will be essential for any disease-modifying therapeutic strategy for Glut1 DS.

Mouse models have made innumerable contributions to understanding the genetic basis of neurological disease and pathogenic mechanisms and to therapy development. Here we consider the current state of mouse genetic models of Developmental and Epileptic Encephalopathy (DEE), representing a set of rare but devastating and largely intractable childhood epilepsies. By examining the range of mouse lines available in this rapidly moving field and by detailing both expected and unusual features in representative examples, we highlight lessons learned in an effort to maximize the full potential of this powerful resource for preclinical studies., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gain-of-function (GOF) variants in K + channels cause severe childhood epilepsies, but there are no mechanisms to explain how increased K + currents lead to network hyperexcitability. Here, we introduce a human Na + -activated K + (K Na ) channel variant (KCNT1-Y796H) into mice and, using a multiplatform approach, find motor cortex hyperexcitability and early-onset seizures, phenotypes strikingly similar to those of human patients. Although the variant increases K Na currents in cortical excitatory and inhibitory neurons, there is an increase in the K Na current across subthreshold voltages only in inhibitory neurons, particularly in those with non-fast-spiking properties, resulting in inhibitory-neuron-specific impairments in excitability and action potential (AP) generation. We further observe evidence of synaptic rewiring, including increases in homotypic synaptic connectivity, accompanied by network hyperexcitability and hypersynchronicity. These findings support inhibitory-neuron-specific mechanisms in mediating the epileptogenic effects of KCNT1 channel GOF, offering cell-type-specific currents and effects as promising targets for therapeutic intervention., Competing Interests: Declaration of Interests S.C. serves a consultant for Q-State Biosciences. D.B.G. is a founder of and holds equity in Praxis, serves as a consultant to AstraZeneca, and has received research support from Janssen, Gilead, Biogen, AstraZeneca, and UCB. M.J.B. has received research support from Janssen. The remaining authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABA A receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Developmental and epileptic encephalopathy (DEE) associated with de novo variants in the gene encoding dynamin-1 (DNM1) is a severe debilitating disease with no pharmacological remedy. Like most genetic DEEs, the majority of DNM1 patients suffer from therapy-resistant seizures and comorbidities such as intellectual disability, developmental delay, and hypotonia. We tested RNAi gene therapy in the Dnm1 fitful mouse model of DEE using a Dnm1-targeted therapeutic microRNA delivered by a self-complementary adeno-associated virus vector. Untreated or control-injected fitful mice have growth delay, severe ataxia, and lethal tonic-clonic seizures by 3 weeks of age. These major impairments are mitigated following a single treatment in newborn mice, along with key underlying cellular features including gliosis, cell death, and aberrant neuronal metabolic activity typically associated with recurrent seizures. Our results underscore the potential for RNAi gene therapy to treat DNM1 disease and other genetic DEEs where treatment would require inhibition of the pathogenic gene product., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mutations in the X-linked gene IQSEC2 are associated with multiple cases of epilepsy, epileptic encephalopathy, intellectual disability and autism spectrum disorder, the mechanistic understanding and successful treatment of which remain a significant challenge in IQSEC2 and related neurodevelopmental genetic diseases. To investigate disease etiology, we studied behaviors and synaptic function in IQSEC2 deficient mice. Hemizygous Iqsec2 null males exhibit growth deficits, hyperambulation and hyperanxiety phenotypes. Adult hemizygotes experience lethal spontaneous seizures, but paradoxically have a significantly increased threshold to electrically induced limbic seizures and relative resistance to chemically induced seizures. Although there are no gross defects in brain morphology, hemizygotes exhibit stark hippocampal reactive astrogliosis. Electrophysiological recordings of hippocampal neurons reveal increased excitatory drive specifically onto interneurons, and significant alterations in intrinsic electrical properties specific to the interneuron population. As they age, hemizygotes also develop an increased abundance of parvalbumin-positive interneurons in the hippocampus, neurons in which IQSEC2 is expressed in addition to the excitatory neurons. These findings point to a novel role of IQSEC2 in hippocampal interneuron synaptic function and development with implications for a class of intractable neurodevelopmental diseases., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

ARFGEF1 encodes a guanine exchange factor involved in intracellular vesicle trafficking, and is a candidate gene for childhood genetic epilepsies. To model ARFGEF1 haploinsufficiency observed in a recent Lennox Gastaut Syndrome patient, we studied a frameshift mutation (Arfgef1 fs ) in mice. Arfgef1 fs/+ pups exhibit signs of developmental delay, and Arfgef1 fs/+ adults have a significantly decreased threshold to induced seizures but do not experience spontaneous seizures. Histologically, the Arfgef1 fs/+ brain exhibits a disruption in the apical lining of the dentate gyrus and altered spine morphology of deep layer neurons. In primary hippocampal neuron culture, dendritic surface and synaptic but not total GABA A receptors (GABA A R) are reduced in Arfgef1 fs/+ neurons with an accompanying decrease in the number of GABA A R-containing recycling endosomes in cell body. Arfgef1 fs/+ neurons also display differences in the relative ratio of Arf6 + :Rab11 + :TrfR + recycling endosomes. Although the GABA A R-containing early endosomes in Arfgef1 fs/+ neurons are comparable to wildtype, Arfgef1 fs/+ neurons show an increase in the number of GABA A R-containing lysosomes in dendrite and cell body. Together, the altered endosome composition and decreased neuronal surface GABA A R results suggests a mechanism whereby impaired neuronal inhibition leads to seizure susceptibility., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Area II of the 2014 Epilepsy Research Benchmarks aims to establish goals for preventing the development and progression of epilepsy. In this review, we will highlight key advances in Area II since the last summary of research progress and opportunities was published in 2016. We also highlight areas of investigation that began to develop before 2016 and in which additional progress has been made more recently.

RNA-protein interaction plays important roles in post-transcriptional regulation. Recent advancements in cross-linking and immunoprecipitation followed by sequencing (CLIP-seq) technologies make it possible to detect the binding peaks of a given RNA binding protein (RBP) at transcriptome scale. However, it is still challenging to predict the functional consequences of RBP binding peaks. In this study, we propose the Protein-RNA Association Strength (PRAS), which integrates the intensities and positions of the binding peaks of RBPs for functional mRNA targets prediction. We illustrate the superiority of PRAS over existing approaches on predicting the functional targets of two related but divergent CELF (CUGBP, ELAV-like factor) RBPs in mouse brain and muscle. We also demonstrate the potential of PRAS for wide adoption by applying it to the enhanced CLIP-seq (eCLIP) datasets of 37 RNA decay related RBPs in two human cell lines. PRAS can be utilized to investigate any RBPs with available CLIP-seq peaks. PRAS is freely available at http://ouyanglab.jax.org/pras/., Competing Interests: The authors have declared that no competing interests exist.

Here we present an open-source R package 'meaRtools' that provides a platform for analyzing neuronal networks recorded on Microelectrode Arrays (MEAs). Cultured neuronal networks monitored with MEAs are now being widely used to characterize in vitro models of neurological disorders and to evaluate pharmaceutical compounds. meaRtools provides core algorithms for MEA spike train analysis, feature extraction, statistical analysis and plotting of multiple MEA recordings with multiple genotypes and treatments. meaRtools functionality covers novel solutions for spike train analysis, including algorithms to assess electrode cross-correlation using the spike train tiling coefficient (STTC), mutual information, synchronized bursts and entropy within cultured wells. Also integrated is a solution to account for bursts variability originating from mixed-cell neuronal cultures. The package provides a statistical platform built specifically for MEA data that can combine multiple MEA recordings and compare extracted features between different genetic models or treatments. We demonstrate the utilization of meaRtools to successfully identify epilepsy-like phenotypes in neuronal networks from Celf4 knockout mice. The package is freely available under the GPL license (GPL> = 3) and is updated frequently on the CRAN web-server repository. The package, along with full documentation can be downloaded from: https://cran.r-project.org/web/packages/meaRtools/., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: DBG reports Equity from Pairnomix and Praxis, personal fees from Eli Lilly (travel honorarium) and AstraZeneca and research grants from Janssen, Gilead, Biogen and UCB. QW declares affiliation with and employment by Simcere Diagnostics Co, Ltd. MAB declares affiliation with and employment by Axion BioSystems. All other authors have declared that no competing interests exist.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease.

Dynamin 1 is a large neuron-specific GTPase involved in the endocytosis and recycling of pre-synaptic membranes and synaptic vesicles. Mutations in the gene encoding dynamin 1 (DNM1) underlie two epileptic encephalopathy syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Mice homozygous for the Dnm1 "fitful" mutation, a non-synonymous coding variant in an alternatively spliced exon of Dnm1 (exon 10a; isoform designation: Dnm1a(Ftfl)) have an epileptic encephalopathy-like disorder including lethal early onset seizures, locomotor and neurosensory deficits. Although fitful heterozygotes have milder recurrent seizures later in life, suggesting an additive or semi-dominant mechanism, the molecular etiology must also consider the fact that Dnm1a(Ftfl) exerts a dominant negative effect on endocytosis in vitro. Another complication is that the fitful mutation induces alterations in the relative abundance of Dnm1 splice variants; mutants have a downregulation of Dnm1a and an upregulation of Dnm1b, changes which may contribute to the epileptic pathology. To examine whether Dnm1a loss of function, Dnm1a(Ftfl) dominance or compensation by Dnm1b is the most critical for severe seizures, we studied alternate isoform-specific mutant mice. Mice lacking Dnm1 exon 10a or Dnm1 exon 10b have neither spontaneous seizures nor other overt abnormalities, suggesting that in normal conditions the major role of each isoform is redundant. However, in the presence of Dnm1a(Ftfl) only exon 10a deleted mice experience severe seizures. These results reveal functional differences between Dnm1a and Dnm1b isoforms in the presence of a challenge, i.e. toxic Dnm1(Ftfl), while reinforcing its effect explicitly in this model of severe pediatric epilepsy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cultured neuronal networks monitored with microelectrode arrays (MEAs) have been used widely to evaluate pharmaceutical compounds for potential neurotoxic effects. A newer application of MEAs has been in the development of in vitro models of neurological disease. Here, we directly evaluated the utility of MEAs to recapitulate in vivo phenotypes of mature microRNA-128 (miR-128) deficiency, which causes fatal seizures in mice. We show that inhibition of miR-128 results in significantly increased neuronal activity in cultured neuronal networks derived from primary mouse cortical neurons. These results support the utility of MEAs in developing in vitro models of neuroexcitability disorders, such as epilepsy, and further suggest that MEAs provide an effective tool for the rapid identification of microRNAs that promote seizures when dysregulated., (© 2016 McSweeney et al.; Published by Cold Spring Harbor Laboratory Press.)

The childhood epileptic encephalopathies (EE's) are seizure disorders that broadly impact development including cognitive, sensory and motor progress with severe consequences and comorbidities. Recently, mutations in DNM1 (dynamin 1) have been implicated in two EE syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Dnm1 encodes dynamin 1, a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Dnm1Ftfl or "fitful" mice carry a spontaneous mutation in the mouse ortholog of DNM1 and recapitulate many of the disease features associated with human DNM1 patients, providing a relevant disease model of human EE's. In order to examine the cellular etiology of seizures and behavioral and neurological comorbidities, we engineered a conditional Dnm1Ftfl mouse model of DNM1 EE. Observations of Dnm1Ftfl/flox mice in combination with various neuronal subpopulation specific cre strains demonstrate unique seizure phenotypes and clear separation of major neurobehavioral comorbidities from severe seizures associated with the germline model. This demonstration of pleiotropy suggests that treating seizures per se may not prevent severe comorbidity observed in EE associated with dynamin-1 mutations, and is likely to have implications for other genetic forms of EE.

Alzheimer's disease (AD) is a leading cause of dementia in the elderly and is characterized by amyloid plaques, neurofibrillary tangles (NFTs) and neuronal dysfunction. Early onset AD (EOAD) is commonly caused by mutations in amyloid precursor protein (APP) or genes involved in the processing of APP including the presenilins (e.g. PSEN1 or PSEN2). In general, mouse models relevant to EOAD recapitulate amyloidosis, show only limited amounts of NFTs and neuronal cell dysfunction and low but significant levels of seizure susceptibility. To investigate the effect of genetic background on these phenotypes, we generated APPswe and PSEN1de9 transgenic mice on the seizure prone inbred strain background, DBA/2J. Previous studies show that the DBA/2J genetic background modifies plaque deposition in the presence of mutant APP but the impact of PSEN1de9 has not been tested. Our study shows that DBA/2J.APPswePSEN1de9 mice are significantly more prone to premature lethality, likely to due to lethal seizures, compared to B6.APPswePSEN1de9 mice-70% of DBA/2J.APPswePSEN1de9 mice die between 2-3 months of age. Of the DBA/2J.APPswePSEN1de9 mice that survived to 6 months of age, plaque deposition was greatly reduced compared to age-matched B6.APPswePSEN1de9 mice. The reduction in plaque deposition appears to be independent of microglia numbers, reactive astrocytosis and complement C5 activity.

Objective: To elucidate the functional consequences of epileptic encephalopathy-causing de novo mutations in DNM1 (A177P, K206N, G359A), which encodes a large mechanochemical GTPase essential for neuronal synaptic vesicle endocytosis., Methods: HeLa and COS-7 cells transfected with wild-type and mutant DNM1 constructs were used for transferrin assays, high-content imaging, colocalization studies, Western blotting, and electron microscopy (EM). EM was also conducted on the brain sections of mice harboring a middle-domain Dnm1 mutation (Dnm1 (Ftfl))., Results: We demonstrate that the expression of each mutant protein decreased endocytosis activity in a dominant-negative manner. One of the G-domain mutations, K206N, decreased protein levels. The G359A mutation, which occurs in the middle domain, disrupted higher-order DNM1 oligomerization. EM of mutant DNM1-transfected HeLa cells and of the Dnm1 (Ftfl) mouse brain revealed vesicle defects, indicating that the mutations likely interfere with DNM1's vesicle scission activity., Conclusion: Together, these data suggest that the dysfunction of vesicle scission during synaptic vesicle endocytosis can lead to serious early-onset epilepsies.

Background: Spike-wave discharges (SWD) found in neuroelectrical recordings are pathognomonic to absence epilepsy. The characteristic spike-wave morphology of the spike-wave complex (SWC) constituents of SWDs can be mathematically described by a subset of possible spectral power and phase values. Morlet wavelet transform (MWT) generates time-frequency representations well-suited to identifying this SWC-associated subset., New Method: MWT decompositions of SWDs reveal spectral power concentrated at harmonic frequencies. The phase relationships underlying SWC morphology were identified by calculating the differences between phase values at SWD fundamental frequency from the 2nd, 3rd, and 4th harmonics, then using the three phase differences as coordinates to generate a density distribution in a {360°×360°×360°} phase difference space. Strain-specific density distributions were generated from SWDs of mice carrying the Gria4, Gabrg2, or Scn8a mutations to determine whether SWC morphological variants reliably mapped to the same regions of the distribution, and if distribution values could be used to detect SWD., Comparison With Existing Methods: To the best of our knowledge, this algorithm is the first to employ spectral phase to quantify SWC morphology, making it possible to computationally distinguish SWC morphological subtypes and detect SWDs., Results/conclusions: Proof-of-concept testing of the SWDfinder algorithm shows: (1) a major pattern of variation in SWC morphology maps to one axis of the phase difference distribution, (2) variability between the strain-specific distributions reflects differences in the proportions of SWC subtypes generated during SWD, and (3) regularities in the spectral power and phase profiles of SWCs can be used to detect waveforms possessing SWC-like morphology., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Absence epilepsy (AE) is a complex, heritable disease characterized by a brief disruption of normal behavior and accompanying spike-wave discharges (SWD) on the electroencephalogram. Only a handful of genes has been definitively associated with AE in humans and rodent models. Most studies suggest that genetic interactions play a large role in the etiology and severity of AE, but mapping and understanding their architecture remains a challenge, requiring new computational approaches. Here we use combined analysis of pleiotropy and epistasis (CAPE) to detect and interpret genetic interactions in a meta-population derived from three C3H × B6J strain crosses, each of which is fixed for a different SWD-causing mutation. Although each mutation causes SWD through a different molecular mechanism, the phenotypes caused by each mutation are exacerbated on the C3H genetic background compared with B6J, suggesting common modifiers. By combining information across two phenotypic measures - SWD duration and frequency - CAPE showed a large, directed genetic network consisting of suppressive and enhancing interactions between loci on 10 chromosomes. These results illustrate the power of CAPE in identifying novel modifier loci and interactions in a complex neurological disease, toward a more comprehensive view of its underlying genetic architecture., (© 2014 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Objective: Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV ) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability, mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity., Methods: We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, a U.S. Food and Drug Administration (FDA)-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2a(Q54) mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting, and survival of Scn2a(Q54) mice., Results: We found that ranolazine was capable of reducing seizure frequency by approximately 50% in Scn2a(Q54) mice. The more potent persistent current blocker GS967 reduced seizure frequency by >90% in Scn2a(Q54) mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2a(Q54) mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2a(Q54) mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting., Significance: Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and that this compound could inform development of new agents., (Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.)

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype - making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era.

In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8a(V929F)). Here, we confirmed the prior suggestion that Scn8a(V929F) exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8a(V929F) is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2(V752F) as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2(V752F) functional analysis revealed an overall gain-of-function and for NaV1.6(V929F) revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2(V752F) due to "functional silencing" of NaV1.6(V929F) suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6(V929F) loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as absence epilepsy., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Twenty-seven inbred strains of mice were tested for spike-wave discharge (SWD) activity by video-electroencephalographic recordings over a 24-h recording period. Eight strains had reproducible, frequent SWDs, including five strains (C57BLKS/J, CBA/J, DBA/1J, NOR/LtJ, SM/J) previously undiagnosed for this distinctive phenotype. Eighteen other strains exhibited no such activity. Spike-wave discharges usually occurred while the subject was motionless, and in a significant number of annotated instances coincided with an arrest of the subject's relatively unrestrained locomotor activity, which resumed immediately after the discharge ended. In all five new strains, SWDs were suppressed by ethosuximide administration. From the genealogy of inbred strains, we suggest that two ancestors, A and DBA, transmitted genotypes required for SWD in all positive strains. Together these strains with SWDs provide new opportunities to understand the genetic core susceptibility of this distinctive electroencephalographic activity and to explore its relationship to absence epilepsy, a human disorder for which few genes are known., (© 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Mutations of MECP2 cause Rett syndrome (RTT), a neurodevelopmental disorder leading to loss of motor and cognitive functions, impaired social interactions, and seizure at young ages. Defects of neuronal circuit development and function are thought to be responsible for the symptoms of RTT. The majority of RTT patients show recurrent seizures, indicating that neuronal hyperexcitation is a common feature of RTT. However, mechanisms underlying hyperexcitation in RTT are poorly understood. Here we show that deletion of Mecp2 from cortical excitatory neurons but not forebrain inhibitory neurons in the mouse leads to spontaneous seizures. Selective deletion of Mecp2 from excitatory but not inhibitory neurons in the forebrain reduces GABAergic transmission in layer 5 pyramidal neurons in the prefrontal and somatosensory cortices. Loss of MeCP2 from cortical excitatory neurons reduces the number of GABAergic synapses in the cortex, and enhances the excitability of layer 5 pyramidal neurons. Using single-cell deletion of Mecp2 in layer 2/3 pyramidal neurons, we show that GABAergic transmission is reduced in neurons without MeCP2, but is normal in neighboring neurons with MeCP2. Together, these results suggest that MeCP2 in cortical excitatory neurons plays a critical role in the regulation of GABAergic transmission and cortical excitability.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.

Id2 is a helix-loop-helix transcription factor essential for normal development, and its expression is dysregulated in many human neurological conditions. Although it is speculated that elevated Id2 levels contribute to the pathogenesis of these disorders, it is unknown whether dysregulated Id2 expression is sufficient to perturb normal brain development or function. Here, we show that mice with elevated Id2 expression during embryonic stages develop microcephaly, and that females in particular are prone to generalized tonic-clonic seizures. Analyses of Id2 transgenic brains indicate that Id2 activity is highly cell context specific: elevated Id2 expression in naive neural stem cells (NSCs) in early neuroepithelium induces apoptosis and loss of NSCs and intermediate progenitors. Activation of Id2 in maturing neuroepithelium results in less severe phenotypes and is accompanied by elevation of G1 cyclin expression and p53 target gene expression. In contrast, activation of Id2 in committed intermediate progenitors has no significant phenotype. Functional analysis with Id2-overexpressing and Id2-null NSCs shows that Id2 negatively regulates NSC self-renewal in vivo, in contrast to previous cell culture experiments. Deletion of p53 function from Id2-transgenic brains rescues apoptosis and results in increased incidence of brain tumors. Furthermore, Id2 overexpression normalizes the increased self-renewal of p53-null NSCs, suggesting that Id2 activates and modulates the p53 pathway in NSCs. Together, these data suggest that elevated Id2 expression in embryonic brains can cause deregulated NSC self-renewal, differentiation, and survival that manifest in multiple neurological outcomes in mature brains, including microcephaly, seizures, and brain tumors., (Copyright © 2013 AlphaMed Press.)

Mice deficient for CELF4, a neuronal RNA-binding protein, have a complex seizure disorder that includes both convulsive and non-convulsive seizures, and is dependent upon Celf4 gene dosage and mouse strain background. It was previously shown that Celf4 is expressed predominantly in excitatory neurons, and that deficiency results in abnormal excitatory synaptic neurotransmission. To examine the physiological and molecular basis of this, we studied Celf4-deficient neurons in brain slices. Assessment of intrinsic properties of layer V cortical pyramidal neurons showed that neurons from mutant heterozygotes and homozygotes have a lower action potential (AP) initiation threshold and a larger AP gain when compared with wild-type neurons. Celf4 mutant neurons also demonstrate an increase in persistent sodium current (I(NaP)) and a hyperpolarizing shift in the voltage dependence of activation. As part of a related study, we find that CELF4 directly binds Scn8a mRNA, encoding sodium channel Na(v)1.6, the primary instigator of AP at the axon initial segment (AIS) and the main carrier of I(NaP). In the present study we find that CELF4 deficiency results in a dramatic elevation in the expression of Na(v)1.6 protein at the AIS in both null and heterozygous neurons. Together these results suggest that activation of Na(v)1.6 plays a crucial role in seizure generation in this complex model of neurological disease.

Background: Transposable element (TE)-derived sequence dominates the landscape of mammalian genomes and can modulate gene function by dysregulating transcription and translation. Our current knowledge of TEs in laboratory mouse strains is limited primarily to those present in the C57BL/6J reference genome, with most mouse TEs being drawn from three distinct classes, namely short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs) and the endogenous retrovirus (ERV) superfamily. Despite their high prevalence, the different genomic and gene properties controlling whether TEs are preferentially purged from, or are retained by, genetic drift or positive selection in mammalian genomes remain poorly defined., Results: Using whole genome sequencing data from 13 classical laboratory and 4 wild-derived mouse inbred strains, we developed a comprehensive catalogue of 103,798 polymorphic TE variants. We employ this extensive data set to characterize TE variants across the Mus lineage, and to infer neutral and selective processes that have acted over 2 million years. Our results indicate that the majority of TE variants are introduced though the male germline and that only a minority of TE variants exert detectable changes in gene expression. However, among genes with differential expression across the strains there are twice as many TE variants identified as being putative causal variants as expected., Conclusions: Most TE variants that cause gene expression changes appear to be purged rapidly by purifying selection. Our findings demonstrate that past TE insertions have often been highly deleterious, and help to prioritize TE variants according to their likely contribution to gene expression or phenotype variation.

RNA-binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA-binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in excitatory neurons, including large pyramidal cells of the cerebral cortex and hippocampus, and it regulates excitatory but not inhibitory neurotransmission. We examined mechanisms underlying neuronal hyperexcitability in Celf4 mutants by identifying CELF4 target mRNAs and assessing their fate in the absence of CELF4 in view of their known functions. CELF4 binds to at least 15%-20% of the transcriptome, with striking specificity for the mRNA 3' untranslated region. CELF4 mRNA targets encode a variety of proteins, many of which are well established in neuron development and function. While the overall abundance of these mRNA targets is often dysregulated in Celf4 deficient mice, the actual expression changes are modest at the steady-state level. In contrast, by examining the transcriptome of polysome fractions and the mRNA distribution along the neuronal cell body-neuropil axis, we found that CELF4 is critical for maintaining mRNA stability and availability for translation. Among biological processes associated with CELF4 targets that accumulate in neuropil of mutants, regulation of synaptic plasticity and transmission are the most prominent. Together with a related study of the impact of CELF4 loss on sodium channel Na(v)1.6 function, we suggest that CELF4 deficiency leads to abnormal neuronal function by combining a specific effect on neuronal excitation with a general impairment of synaptic transmission. These results also expand our understanding of the vital roles RNA-binding proteins play in regulating and shaping the activity of neural circuits., Competing Interests: The authors have declared that no competing interests exist.

Mice deficient for the gene encoding the RNA-binding protein CELF4 (CUGBP, ELAV-like family member 4) have a complex seizure phenotype that includes both convulsive and non-convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adults. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at the age of 7 weeks. This timing is in contrast to absence-like non-convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch-clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron-specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice., (© 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

Cortico-thalamo-cortical circuits mediate sensation and generate neural network oscillations associated with slow-wave sleep and various epilepsies. Cortical input to sensory thalamus is thought to mainly evoke feed-forward synaptic inhibition of thalamocortical (TC) cells via reticular thalamic nucleus (nRT) neurons, especially during oscillations. This relies on a stronger synaptic strength in the cortico-nRT pathway than in the cortico-TC pathway, allowing the feed-forward inhibition of TC cells to overcome direct cortico-TC excitation. We found a systemic and specific reduction in strength in GluA4-deficient (Gria4(-/-)) mice of one excitatory synapse of the rhythmogenic cortico-thalamo-cortical system, the cortico-nRT projection, and observed that the oscillations could still be initiated by cortical inputs via the cortico-TC-nRT-TC pathway. These results reveal a previously unknown mode of cortico-thalamo-cortical transmission, bypassing direct cortico-nRT excitation, and describe a mechanism for pathological oscillation generation. This mode could be active under other circumstances, representing a previously unknown channel of cortico-thalamo-cortical information processing.