The last decade has witnessed an unprecedented expansion of the epilepsy genome that is providing a fundamentally new understanding of defective molecular and cellular programs in the developing epileptic brain. This progress is due to transformative changes both in the technology applied to gene variant detection in humans, and the tools needed to engineer and control these genes in the nervous system. The result has driven an explosion of new biology that accompanies each of the novel variants as they are analyzed in experimental genetic models in human cells, mice, zebrafish, and flies. The chapters in this section capture some, but certainly not all, of the excitement, information, and technology development using these novel strategies to investigate how gene errors destabilize cortical networks.

Human epilepsy gene discovery has turned from the slow but steady pace over the last two decades that began with the isolation of the first familial inherited epilepsy genes from informative pedigrees with multiple affected individuals, to enter the fast lane of whole exome de novo variant isolation in pediatric epilepsy singleton probands. This approach is now contributing rare variants in both known and unknown genes that are promptly translated into preclinical models. An inescapable observation regarding this transition has been the shift in the biology of these genes from almost exclusively ion channel based in the familial cases to a far broader ontology in the de novo cases, implicating pathways governing membrane and synaptic excitability and neurotransmitter homeostasis, and even more distant pathways that in some way regulate both the firing and wiring of the developing cortex. In Chapter 41, Bonkowski and Mefford describe the advances in next generation sequencing of various epilepsy syndromes by interrogating both exome sequences (~1% of the genome) as well as variants lying outside protein coding regions.

Once detected, each variant requires functional validation, and this is most accurately accomplished within the context of the patient’s own genome using patient-derived cells. In Chapter 42, Varela, Samarasinghe, and Parent describe the importance and utility of this approach in IPSC-derived models of cell cultures and organoids. Somatic mutations that underlie focal epilepsies and cortical malformations are now frequently discovered in resected human tissue and are also best studied in the context of the individual’s own genomic environment. In Chapter 43, Ribierre and Baulac illustrate the search for somatic mutations in genes in the mTOR and galactose transporter pathways and their functional assessment in organoids.

Among the ion channel gene family, sodium channelopathy was historically the sentinel discovery underlying several clinical syndromes of human inherited epilepsy and has led the way from molecular diagnosis to mechanistic scrutiny in a variety of prototypical experimental neurogenetic models. In Chapter 44, Yamakawa, Meisler, and Isom, pioneer laboratories in this field, recount the complexities of electrophysiological analysis in mouse conditional models of several different sodium channel subunits that provide the basis for advancing these genes to the translational stage. Potassium channelopathy, actually the first channel gene to be discovered in a spontaneous experimental model, the Drosophila mutant shaker, has rapidly caught up in the race to understand how gain and loss-of-function variants can both give rise to epilepsy. In Chapter 45, Weston and Tzingounis navigate the landscape of epileptogenic variants in several potassium channel subtypes and propose solutions to these paradoxical outcomes. Likewise, calcium channelopathy, initially discovered in spontaneous mouse mutants, is a forerunner of over 20 genes now linked to childhood absence epilepsy in monogenic mouse models. These genes have raised a host of issues related to the complex role of these high- and low-voltage activated channels in excitability, exocytosis, and gene regulation. In Chapter 46, Rossignol provides an extensive accounting of the diverse pathophysiology and spectrum of clinical phenotypes resulting from gain-of-function and loss-of-function mutations in an important subset of high-voltage activated calcium channels.

In each of these channelopathies, and certainly in countless other monogenic epilepsies, detailed analysis in mouse models reveals an inordinate impact on interneuronal function. Since defective inhibition is a prime suspect in all epileptic networks, the remaining chapters in this section delve into new insights on mechanisms interfering with the birth of interneurons and their dysmaturation. In Chapter 47, Pai, Vogt, Hu, and Rubenstein provide a comprehensive review of the birth, migration, and fate of GABAergic neurons, as well as recent discoveries at the transcriptional level guiding interneuron development. In Chapter 48, Olsen, Wallner, and Rogawski review the recent advances in GABA receptor structural biology, including high-resolution cryo-electron microscopy of purified recombinantly expressed heterotrimeric GABAR subunits, which reveal receptor structure at an unprecedented level of detail, enabling definition of the molecular binding sites GABAR-targeted drugs. This achievement marks the “end of the beginning” of translating the regulation of these receptors into therapies for seizures and their comorbidities. Finally, along these lines, in Chapter 49, Kabow and Mahoney discuss emerging analytical tools and insights into the epigenetic “cross-talk” regulating the transcriptional profiles of thousands of genes responsible for developing and maintaining the epileptogenic state.

Although not explored in this section, it is important to recognize that the ever-expanding complexity of neurons and their connectome revealed by high-resolution molecular tools over the past decade now demands major conceptual changes in our current approach to understanding the epileptic brain. We are now shifting from the analysis of single genes with a known function to the realization that each of them combines into multigenic pathways that lead to distinct epilepsy phenotypes. Thus, the heterogeneity index of genotype: phenotype continues to rise as we uncover the many levels of biology that different variants in a single gene can engage. New tools such as ATAC-seq that assess open chromatin regions and TRAP that selectively label active gene transcription in specific neurons can pinpoint the onset of downstream molecular lesions, while targeted CRISPR genome editing and optogenetic strategies facilitate epileptic network dissection. Some genes for epilepsy are selectively expressed in specific cell types; others are diffusely transcribed but into different cell-type specific isoforms. Rather than modeling a single type of inhibitory neuron, we now appreciate that over 40 entirely distinct anatomical and transcriptomic examples of these cells balance inhibition and excitation in critical networks, and that a host of genes regulate the strength of their functional connectivity. Cellular plasticity, particularly during development, is also remarkably complex: some cells remain molecularly stable over long periods, while others are more sensitive to synaptic activity. The same is true for their synaptic connectivity and ability to rewire key microcircuits.

A further challenge for the future is illustrated by a new understanding of aggregate neuronal behavior during seizures. Recent evidence (Chapter 21 by Meyer and Maheshwari) obtained using two-photon imaging of generalized spike-wave seizure activity in an absence epilepsy mouse model indicates that neuronal participation in thalamocortical seizures is not hard-wired, where every excitatory neuron in the neocortex fires simultaneously followed by the lockstep activation of all interneurons, as might have been predicted from this hypersynchronous EEG discharge pattern. Instead, many superficial cortical neurons are recruited into the bursting network in a nearly stochastic fashion, sometimes bursting during one seizure and silent during the next. Some interneurons may even activate before the seizure starts. These dynamic multicellular population observations challenge us to develop more realistic network models as opposed to hard-wired multiplex NEURON models built of static unicellular participants. Armed with these evolving concepts and tools, and in keeping with the goals of the Jasper research mission, we can envision the next edition addressing three pressing questions that arise from gene models. The first question is when does inherited epileptogenesis actually begin? The preclinical incubation period clearly varies for each gene, since postnatal seizure onset is highly variable. The second question is whether there is a common critical time window for reversal. Finally, for each gene, which other genes strongly affect its phenotype? It is now clear that co-mutation of even a single gene can either mask or potentiate the clinical phenotype. Expanding this epistatic profile for common monogenic epilepsies may lead to novel targets for pharmacoresistant epilepsies.