Jeffrey L. Noebels - US grants

imaging /visualization /scanning; nervous system; histology; biomedical facility; mental retardation; electron microscopy; in situ hybridization; histochemistry /cytochemistry; immunocytochemistry;

Neurosensory evaluation of defined gene mutations in inbred strains of mice is an essential component of the genetic analysis of brain and behavior, and a critical tool in assessing responses to pharmacological treatment. The specific aims of this project are to develop and implement rapid, high-throughput electrophysiologic survey methods and an open electronic reference database containing standardized neurophysiological evaluation of inbred strains and mutants, including surface cortical EEG, depth-recorded hippocampal EEG, visual evoked potentials, and brainstem auditory evoked potentials, in awake and behaving mice. The specific aims include: 1) A technological component, involving development of lighter-weight, higher- electrode density epidural and depth electrode arrays for chronic implantation. Current techniques will be enhanced by implementation of non-traumatic, flexible zero-insertion force microconnectors and calibrated, region-specific placement. Electrophysiological data will be acquired digitally, combined with split-screen digital video, and programs to rapidly display and share online over the internet will be implemented. 2) A quantitative data analysis component, involving application of spectral and topological analysis of spontaneous electrocorticograms and averaged evoked potentials to catalog inbred strains. 3) A phenotypic screening standards component: Multi-parametric studies will be performed to assess variability related to diurnal rhythm, time of day, and repeated sampling; age and behavioral state; and characterization of technical artifacts in order to optimize reproducibility and validation of all basal and evoked data samples. 4) An instructional component: all technqiues will be standardized and methods for quantitative analysis and screening will be fully detailed and disseminated for adoption by all NIH mutagenesis centers and other laboratories. 5) A scientific database and informatics component: Storage and retrieval of all raw and analyzed data on inbred strains will be deposited in an open reference database accessible from a dedicated website over the Internet, and summary results published. Following validation, data on specific mutants screened for individual investigators will be available by consent shortly after study. The reference data on inbred strains and defined mutants from multiple centers will be initiated in a common access database, allowing laboratories to subsequently deposit, retrieve, and even reanalyze reference data from mice screened by all centers. Hyperlinks will be created with other databases. This project will provide the framework for normative data to be used for comparative electrophysiological, behavioral and experimental pharmacology on inbred mouse strains and defined gene mutations.

Neurosensory evaluation of defined gene mutations in inbred strains of mice is an essential component of the genetic analysis of brain and behavior, and a critical tool in assessing responses to pharmacological treatment. The specific aims of this project are to develop and implement rapid, high-throughput electrophysiologic survey methods and an open electronic reference database containing standardized neurophysiological evaluation of inbred strains and mutants, including surface cortical EEG, depth-recorded hippocampal EEG, visual evoked potentials, and brainstem auditory evoked potentials, in awake and behaving mice. The specific aims include: 1) A technological component, involving development of lighter-weight, higher- electrode density epidural and depth electrode arrays for chronic implantation. Current techniques will be enhanced by implementation of non-traumatic, flexible zero-insertion force microconnectors and calibrated, region-specific placement. Electrophysiological data will be acquired digitally, combined with split-screen digital video, and programs to rapidly display and share online over the internet will be implemented. 2) A quantitative data analysis component, involving application of spectral and topological analysis of spontaneous electrocorticograms and averaged evoked potentials to catalog inbred strains. 3) A phenotypic screening standards component: Multi-parametric studies will be performed to assess variability related to diurnal rhythm, time of day, and repeated sampling; age and behavioral state; and characterization of technical artifacts in order to optimize reproducibility and validation of all basal and evoked data samples. 4) An instructional component: all technqiues will be standardized and methods for quantitative analysis and screening will be fully detailed and disseminated for adoption by all NIH mutagenesis centers and other laboratories. 5) A scientific database and informatics component: Storage and retrieval of all raw and analyzed data on inbred strains will be deposited in an open reference database accessible from a dedicated website over the Internet, and summary results published. Following validation, data on specific mutants screened for individual investigators will be available by consent shortly after study. The reference data on inbred strains and defined mutants from multiple centers will be initiated in a common access database, allowing laboratories to subsequently deposit, retrieve, and even reanalyze reference data from mice screened by all centers. Hyperlinks will be created with other databases. This project will provide the framework for normative data to be used for comparative electrophysiological, behavioral and experimental pharmacology on inbred mouse strains and defined gene mutations.

DESCRIPTION (provided by applicant): Our long term goal is to learn how an inherited gene error produces a specific pattern of epilepsy in the developing brain, and to provide an exact description of subsequent seizure-induced plasticity within affected neural networks. Generalized absence seizures of the spike-wave pattern comprise a major category of inherited epilepsy in children. Although genes for this phenotype are known in mice, the underlying basic neuronal excitability mechanisms, their degree of overlap, and the effects of spike-wave hypersynchrony on developing neural circuitry have not been clearly defined. The primary goal of this project is to examine specific network abnormalities identified in isolated thalamocortical brain slices of stargazer (gamma2) mutant mice, and compare them to 3 other mutants of voltage-paled calcium channel subunits, tottering (alpha1A), lethargic (beta4), and ducky (alpha2delta2), all with gene-linked spike-wave seizures. The stargazer gene product gamma2 (stargazin), has homology to the gamma1 subunit of the muscle voltage-gated calcium channel, and interacts with neuronal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors. In the previous project period, we identified specific excitability defects in cortical, thalamic, and hippocampal neurons in these mutants. We hypothesize: (1) that spike-wave discharges in these mutant mice arise from multiple distinct thalamocortical network excitability defects that alter membrane currents, synaptic transmission, and gene expression, and (2) that inhibitory and excitatory synapses are differentially affected by these channelopathies. Using slice patch clamp, optical recordings, molecular anatomy, and transgenic methods to study the mutant neurons, we will test specific hypotheses regarding the membrane and synaptic mechanisms underlying this network defect. We will explore the functional role of the gene-linked defects in epileptogenesis by determining which components arise developmentally as a primary cellular expression of the channelopathy, and which arise secondarily as a product of seizure-induced neuroplasticity. In specific aim 1, we will analyze intrinsic membrane properties and calcium (Ca)2+ currents of control and mutant thalamic neurons to quantify changes in burst firing properties linked to each genotype. We will explore the basis for T-type current alterations in non-T-type gene mutants. In specific aim 2, we will examine the role of specific calcium channel subunits in neurotransmitter release from presynaptic terminals of mutant cortical neurons. In specific aim 3, we will explore the molecular basis for an abnormal response to glutamate receptor antagonists in stargazer cortical circuits. In specific aim 4 we will use transgenic rescue strategies to dissect the role of inhibitory brain pathways in the mutant phenotype. In each specific aim, we will examine the development of these properties relative to the postnatal onset of spike-wave epilepsy. These studies will directly test key hypotheses concerning basic mechanisms of thalamocortical oscillations produced by calcium ion channelopathies, and the degree of long-term cellular and molecular neuroplasticity that may accompany early seizures of the spike-wave pattern.

DESCRIPTION (provided by applicant): Ion channel genes represent 1.5% of the human genome, and inherited mutations of these genes elicit a diverse array of clinical disorders of brain, nerve, muscle and heart. In brain, single gene channelopathies are the predominant cause (13/14) of rare mendelian idiopathic epilepsy syndromes, but their contribution to common sporadic epilepsy is unknown. High rates of de novo mutation and complex polygenic inheritance (the "common disease-common variant" model) are two attractive explanations for the role of ion channel variation in sporadic cases. Together with their important pathogenic role, ion channels are also the primary molecular targets of most antiepileptic drugs, and genetic variation in channel subunits may independently contribute to pharmacoresistance. This project combines basic and clinical research on ion channelopathy and specific epilepsy phenotypes with the large scale gene sequencing capacity and mutation analysis resources of the Baylor Human Genome Sequencing Center in order to test the general hypothesis that profiling the coding sequences of large numbers of channel genes in individual epilepsy patients can reveal novel mutations and patterns of common allelic variants that determine epilepsy susceptibility and pharmacoresistance. We will complete the development and optimization of a multiplex primer array that allows rapid and scalable parallel exon sequencing of 100 candidate ion channel genes in 500 patients with specific clinical epilepsy phenotypes and in 500 ethnically-matched controls. A public database of human ion channel gene variation will be generated to facilitate data-sharing. These data will be used in two ways. First, the biophysical and pharmacological properties of a subset of channel gene polymorphisms with predicted protein coding variation will be analyzed in mammalian expression systems in order to define a validated subset of functional gene variants of human ion channels relevant to epilepsy. This list is essential to examine models relating specific pathophysiological properties of ion channels to the patterns associated with epilepsy. Second, the sequence of the 100 channel genes will be assembled into a profile of each individual (their "channotype") and used to test the statistical association of different channotypes with epilepsy phenotypes. Preliminary analysis of all exons of 7 channel genes in 50 patients and 50 controls has detected novel and previously reported SNPs (coding and non-coding) and microdeletions, validating the efficiency of the data collection pipeline. Using robotic processing and automated mutation detection algorithms, we will scale the number of genes and patients to attain the statistical power to address the channotype-phenotype association hypotheses. The associations identified in this study will address a major hypothesis underlying the complex genetics of epilepsy, accelerate development of individualized clinical risk assessments for epilepsy, and examine a novel mechanism of resistance to antiepileptic drugs in children and adults with common idiopathic forms of the disorder.

[unreadable] DESCRIPTION (provided by applicant): This proposal is for planning support to establish a new neurobiology of disease course and track of graduate study at Baylor College of Medicine (BCM). BCM has a long history of excellence in neuroscience research - both basic and clinical. This program represents a new effort to develop an integrated course and training track in graduate education that draws on the strengths of faculty from 10 basic and clinical departments, providing a formal course that includes a detailed first principles approach to the study of the underlying cellular and molecular processes of diseases that affect the nervous system throughout the lifespan. The collaborative effort approaches the study of the nervous system from the perspective of rigorous scientific analysis of the pathobiology of disorders from the realms of Neurology, Neurosurgery, Pediatrics, Psychiatry and the special senses (Ophthalmology and Otolaryngology). The program will draw students from existing graduate programs in Neuroscience (currently supported by an NIH NIGMS Dr. Ruth L. Kirschstein NRSA T32 training grant that is funded until 2009), the M.D./Ph.D. program and the newly formed Translational Biology and Molecular Medicine graduate training program at Baylor College of Medicine, as well as providing access to the neurobiology of diseases course to other interested students from graduate programs in molecular and cellular biology, developmental biology and genetics. The course will include team lectures from basic and clinical neuroscientists as well as exposure to clinical cases through rounds and visits to neuroscience clinical diagnostic and therapeutic procedures. In addition, the course will serve as a standard component of the existing Neuroscience graduate curriculum for all neuroscience students as well as providing a focal point for students who plan to focus their research training primarily in neurobiology of disease. This initiative is being developed at a particularly auspicious time at BCM, in conjunction with the launching of a major strategic plan that includes translational neuroscience, the hiring of a new Chair of Neuroscience, the appointment of the College's first Director for coordinating Neuroscience Initiatives between basic and clinical departments and expanded graduate training initiatives in translational biology. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our long term goal is to learn how an inherited gene error produces a specific pattern of epilepsy in the developing brain, to provide an exact description of seizure-induced plasticity within affected neural networks, and in this project period, to genetically dissect the intervening candidate networks and mechanisms using selective mutant gene expression in mouse models. Spike-wave (SW) absence seizures comprise a major category of inherited epilepsy in children. Mutant genes for this phenotype are known, and their effects on ion channel behavior and routes of convergence on downstream neuronal excitability in pacemaking circuitry are beginning to be clearly defined. We have detected a critical pathway converging on the elevation of thalamic T-type calcium currents, and further showed that isolated T-type channel overexpression in wild type mice promotes cortical SW discharges, illuminating a complex but shared plasticity pathway triggered by these genes. We now seek further definition of the precise timing, specific synaptic circuitry, and transcriptional mechanisms mediating inherited P/Q channel-linked network excitability defects in order to determine the reversability of these phenotypes. In specific aim 1, new information from a conditional Cacna1a allele indicates that mice display SW epilepsy even when the P/Q type calcium current defects are engineered to appear with a delayed onset in the third postnatal week. This indicates that aberrant adult firing, not embryonic wiring, is a sufficient cause for this seizure phenotype, thereby demonstrating an important postnatal window of therapeutic opportunity. In specific aims 2 and 3, we will narrow the circuitry required for inherited SW seizures by genetically ablating the P/Q channel gene in other subsets of neurons to determine whether these limbs of the thalamocortical loop are necessary or sufficient for SW phenotypes. In specific aim 4 we will explore transcriptional mechanisms underlying the plasticity of thalamic t-type calcium currents. Since these currents are potentiated by a broad spectrum of neuronal injury, clarifying the mechanism underlying downstream calcium channelopathy remodeling is of central interest in understanding how to prevent or reverse this common form of inherited epilepsy. PUBLIC HEALTH RELEVANCE: This project will determine how a mutation of a single gene causes a specific pattern of epilepsy in the brain, which brain circuits are involved, and when it appears during brain development. An inherited mechanism underlying the pathological transformation of thalamocortical network firing from normal rhythms to abnormal burst firing patterns during absence seizures will be isolated. These findings may lead to novel treatments for a common form of childhood epilepsy.

DESCRIPTION (provided by applicant): Unexplained collapse of cardiac and respiratory rhythmicity is a final common mechanism for SUDEP, a major and preventable cause of death in persons with epilepsy. Recent evidence shows that dysfunctional ion channels and receptors co-expressed in brain, autonomic, heart, and respiratory pathways, along with clinical measures of functional disturbances in these pathways at times surrounding seizures represent detectable and potentially treatable risk factors for SUDEP. This proposal describes an integrated multicenter and multidisciplinary collaborative project that will combine a basic science, human neurogenetics, and clinical physiology approaches to these biological risk factors in a bench to bedside translational research program to identify, validate, and clinically evaluate predictive biomarkers and preventative treatments for SUDEP. The SUDEP Center Research Pipeline will consist of a serially interrelated work flow among 6 investigators in the center. Project 1 (Baylor) will expand the repository of DNA samples from patient at 3 centers (EMU, Dravet Syndrome Clinic, SUDEP DNA Repository) and other national networks which will be analyzed using chip microarrays for >247 prioritized ion channel and receptor genes mediating cardiac arrhythmias, respiratory depression and epilepsy. Projects 2-4 (Baylor U. Michigan, U. Iowa) analyze the biology, physiology, and pharmacology of these and related gene mutations at the cellular and in vivo level in SUDEP mouse models and induced pluripotent stem cells from Dravet Syndrome cases in order to understand and validate the SUDEP phenotypes. Project 5 (U.C. Davis/Childrens Memorial Chicago) will refine clinical respiratory and cardiac biomarkers obtained during epilepsy monitoring of individuals with Dravet Syndrome and others at high risk of sudden death (ictal hypoxemia, cardiac arrhythmia). Once validated, genes from these cases are added to an incremental diagnostic chip in development at Baylor for routine patient risk assessment in clinics in individuals with other clinical biomarkers. PUBLIC HEALTH RELEVANCE: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of premature mortality in idiopathic epilepsy. Preventing SUDEP depends upon identifying biologically predictive risk factors in individuals with epilepsy and using them to mak appropriate therapeutic interventions. The goal of this program is to validate a combined genetic/clinical SUDEP risk profile to screen and treat individuals with epilepsy. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of th individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

? DESCRIPTION (provided by applicant): The primary mission of the SRA is to understand the basic biological mechanisms, genetic and physiological risk factors, predictive diagnostic biomarkers, and potential methods of preventing sudden unexpected death, the most common cause of premature mortality in human epilepsy. This project will test the principal hypothesis that mutation of single genes co-expressed in brain, heart, and brainstem central autonomic neurons increase SUDEP risk by promoting cardiorespiratory arrhythmias. We will identify and validate novel ictal bradycardia and SUDEP gene candidates in mouse models to expand the SUDEP risk genome, and use modifier genes that suppress this risk in forebrain and brainstem autonomic circuits to genetically dissect critical SUDEP pathways. We will examine molecular excitability mechanisms perturbed by these genes in brain and heart, and the role of seizures and hypoxia in pathological network depolarization. We will also explore translational pharmacologic and genetic rescue strategies in these models. These findings will contribute to future genetic risk profiling for SUDEP in clinical exomes and point to new gene-directed approaches to reduce SUDEP in patients at high risk.

? DESCRIPTION (provided by applicant): Epilepsy is a prototypical neural circuit disorder with a one-year prevalence of ~7/1,000 and high cost to society. Genetic or acquired etiologies of epilepsy lead to neuronal circuit hyper-synchrony that manifests as a seizure. A major unsolved question in epilepsy is how single units get recruited, in vivo, during the evolution of seizure events. Specifically it is not known whether neurons fire in a stereotyped pattern or sequence per seizure event, whether this happens reliably or whether/how it depends on cell type. It is important to determine whether pyramidal neurons show different patterns of recruitment than various classes of interneurons, and whether there exist special (hub) units that are reliably engaged and may therefore play an important role in recruiting other units to seizure events. The stargazer mouse is a validated experimental model for human absence epilepsy. Mutation of the protein stargazin leads to impaired AMPA receptor membrane trafficking, and this is thought to suppress primarily excitatory inputs projecting on inhibitory (Parvalbumin+) interneurons (Maheshwari et al., Frontiers in Cellular Neuroscience, 2013). This relative silencing of inhibition is thought to disinhibit the surrounding microcircuit, promoting hyper-synchrony and seizures. Whether this happens in vivo and how it entrains neocortical circuits remains unknown. The role that other interneuronal classes play remains also obscure. We will use chronic two-photon imaging to map how individual cortical neurons are recruited in vivo during stargazer absence seizure events, and to measure their temporal activity profiles and reliability of recruitment. Preliminary data suggests that recruitment is not random, but potentially depends on cell type, laminar location, and the neuron's hub status. Identifying groups of cells that exhibit high levels of synchrony will reveal local sub-networks important for seizure manifestation. Finally, in vivo whole-cell patch clamp experiments will be performed to 1) validate the two photon results, and 2) to study how inhibitory and excitatory inputs evolve during absence seizure events in pyramidal neurons versus in select classes of GABA-ergic interneurons. Optogenetic manipulation of Parvalbumin+ interneuron activity levels will establish causality. Obtained insights into epileptic circuit malfunction will potentially lead to new strategies for cell-targeted therapeutic interventions.

Glioblastoma cells trigger pharmacoresistant seizures that may promote tumor growth and diminish the quality of remaining life. To define the relationship between growth of glial tumors and their neuronal microenvironment, and to identify genomic biomarkers and mechanisms that may point to better prognosis and treatment of drug resistant epilepsy in brain cancer, we are analyzing a new generation of genetically defined CRISPR/in utero electroporation inborn glioblastoma (GBM) tumor models engineered in mice. The molecular pathophysiology of glioblastoma cells and surrounding neurons and untransformed astrocytes will be compared at serial stages of tumor development in three genetic mouse strains: wild type, seizure prone, and seizure resistant. Preliminary data reveal that epileptiform EEG spiking is a very early and reliable preclinical signature of GBM expansion preceding other neurological deficits in these mice, followed by rapidly progressive seizures and death within weeks. Transcriptomic analysis of cortical astrocytes reveals the expansion of a subgroup enriched in pro-synaptogenic genes that may drive hyperexcitability, a novel mechanism of epileptogenesis. In Specific Aim 1 we will systematically define the earliest appearance of cortical hyperexcitability in wild type mice with a prototypical GBM and correlate its progression with in vivo and neuropathological imaging of invasive tumor cell location, in vitro electrophysiology, and molecular markers of key epilepsy pathogenic cascades in peritumoral neurons, including impaired glutamate reuptake, altered GABA gated-chloride gradients, and synaptic densities. In Specific Aim 2 we will correlate these findings with detailed FACS-sorted transcriptomic profiles of both transformed and wild type astrocytes in the peritumoral region to test the novel hypothesis that peritumoral hyperexcitability is driven in part by astrocytic subtypes that disrupt synaptic E/I homeostasis. In Specific Aim 3, we will use this benchmark approach in WT brain to compare growth, electrophysiological and molecular pathological profiles of the same tumor generated in a hyperexcitable brain bearing a single gene deletion (Kcna1) that dramatically lowers the threshold for seizures and shortens lifespan, and in a monogenic deletion strain (MapT/tau) that raises cortical seizure threshold and prolongs life, in order to examine the contribution of host neuronal excitability to tumor expansion. Our approach sets the stage to broadly explore the developmental biology of personalized tumor/host interactions in mice engineered with novel human tumor mutations in specified glial cell lineages.

Abstract Epilepsy affects over 2 million of people in the United States, causing significant morbidity with a high cost to society. While the behavioral and electrophysiological correlates of seizures in patients and animal models have been studied for over a century, the underlying circuit abnormalities are still being elucidated. Generalized spike-wave (SW) absence sei- zures are the most common seizure disorder in children and thought to be exclusively of genetic origin. While over 20 genes are discovered and studied in SW epilepsies, it is still unclear how each genetic lesion impairs normal circuit devel- opment and ultimately results in a seizure-prone cortical circuit. Since the SW seizure phenotype can be very similar de- spite disparate genetic etiologies, a stereotypical circuit deficit may exist which underlies the expression of this seizure pattern. More recently, as we have begun to understand the wiring principles of cortical microcircuits at the level of cell types, it has become possible to ask how disruption of these canonical networks may be responsible for initiating seizure activity and impairing cognitive functions in SW epilepsies, whether the pathogenic circuit changes overlap despite dis- parate molecular lesions, and how a seizure-prone circuit emerges from inherited molecular defects to favor seizure on- set at predictable developmental time-points. This information not only suggests novel and broadly-applicable therapeu- tic targets, but also leads to valuable insights into the functional roles of distinct cell types and specific connectivity principles in normal brain. To answer these important questions, we are taking advantage of three mouse models of ab- sence epilepsy, stargazer, tottering and Gabrg2 mutant mice, which harbor mutations in three unrelated genes but share the same SW phenotype, and propose a comprehensive microcircuit comparison among distinct genotypes at the level of cell types and their connections. We perform a large-scale circuit analysis across a whole column of the somatosen- sory cortex (S1) in three models along the seizure development, by leveraging a high-throughput multi-patching method (up to 12-patch) we recently developed. We will measure multiple neuronal features of distinct cell types within the S1 epileptic circuit, with an emphasis on connectivity and morphology of major groups of cortical GABAergic interneurons. In parallel, the same analysis will be performed on WT littermates as controls to reveal cell type-specific connectivity changes as a function of the genotype and developmental stage. These comprehensive, dynamic comparisons, based on large-scale circuit analyses with sensitive, state-of-the-art methods, will reveal the full extent of abnormal microcircuit structure and functions that are closely associated with seizure onset. Our preliminary data uncover several connectivity defects in these models. The most striking is that stereotypical connectivity and morphology of somatostatin-expressing Martinotti cells are severely disrupted, and this disruption appears to emerge only after seizure onset and is shared by models, suggesting a common circuit deficit underlying absence epilepsy. The potential causative circuit mechanisms will be further tested via network modeling and an in vivo chemogenetic assay. Identification of causative circuit deficits gen- eralized across genetically heterogeneous, yet highly stereotyped SW seizures will direct the field toward the develop- ment of innovative, broadly applicable circuit-based interventions for absence epilepsy and its related comorbidities.

Abstract Our long term goal is to learn how an inherited single gene error produces a specific pattern of epilepsy in the developing brain, to provide an exact description of relevant plasticity within affected neural networks, and to reverse the seizure phenotype at the earliest possible stage. Spike-wave (SW) absence seizures comprise a major category of inherited epilepsy in children, and often herald cognitive deficits and more severe seizures. Over 20 mutant genes for this phenotype are known, and their effects on channel behavior and routes of convergence on excitability within thalamocortical pacemaking circuitry are now more clearly defined. The P/Q calcium channel mouse mutant is a prototype for this analysis, and like other models, shows elevated thalamic T-type calcium currents that are sufficient to generate absence epilepsy, illuminating a shared downstream plasticity pathway triggered by functionally disparate upstream SW genes. The mechanism underlying T-type current remodeling is not understood. In the past project period we narrowed the critical pathogenic microcircuitry and found that selective ablation of P/Q type calcium channels in Layer 6 corticothalamic neurons alone are sufficient to elevate thalamic T currents and cause SW epilepsy, reducing the analysis from the entire brain to a single thalamic afferent synapse, and showed that adult P/Q channel deletion reproduces the childhood syndrome albeit through an alternative pattern of T current circuit remodeling. Using newly created models, we will 1) analyze native and alternative thalamic transcriptome changes to define the molecular basis for T current plasticity in thalamic excitatory and inhibitory neurons and uncover novel epistatic genes participating in this switch, 2) test the thalamic current imbalance in a digenic model to simulate the combinatorial effects of common human CAE variants in T currents, and 3) determine whether we can reverse the T current imbalance and epileptic phenotype in PQ channel mutants by restoring normal PQ function after the onset of seizures. This analysis brings us closer to molecular level treatments of pathogenic gene expression in epilepsy.