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Background: Early preterm (<34 weeks of gestation) small for gestational age infants (<10th percentile birth weight for sex and gestational age) experience high rates of morbidity and mortality, the causes of which are poorly understood. Mounting evidence suggests that genetic disorders contribute. Scarce data exist regarding the prevalence of genetic disorders and their contribution to morbidity and mortality., Objective: This study aimed to determine the proportion of genetic disorders in early preterm small for gestational age infants (with and without congenital anomalies) compared to early preterm appropriate for gestational age infants and the association of genetic disorders with morbidity or mortality., Study Design: This is a retrospective cohort study of infants delivered at 23 and 0/7 to 33 and 6/7 weeks of gestation from 2000 to 2020 from the Pediatrix Clinical Data Warehouse. Data included diagnosed genetic disorders and congenital anomalies, baseline characteristics, and morbidity or mortality. We excluded cases of death in the delivery room before neonatal intensive care unit admission, multiple gestations, and cases transferred after birth or before death or discharge., Results: We identified 223,431 early preterm infants, including 21,180 small for gestational age. Genetic disorders were present in 441 (2.3%) of small for gestational age infants without congenital anomalies, in 194 (10.8%) of small for gestational age infants with congenital anomalies, and in 304 (4.5%) of small for gestational age infants that experienced morbidity or mortality (with or without congenital anomalies). Trisomies 13, 18, and 21 were the most prevalent genetic disorders in these groups, together accounting for 145 small for gestational age infants without congenital anomalies, 117 small for gestational age infants with congenital anomalies, and 166 small for gestational age infants with morbidity or mortality (with or without congenital anomalies). Less prevalent genetic disorders consisted of other aneuploidy (45, X and 47, XXY), copy number variants (13q14 deletion syndrome, cri du chat syndrome, DiGeorge syndrome), and single gene disorders (cystic fibrosis, Fanconi anemia, glucose-6-phosphate dehydrogenase deficiency, hemophilia, hypophosphatasia, sickle cell disease, and thalassemia). Comparatively, genetic disorders were found in 1792 (1.0%) appropriate for gestational age infants without congenital anomalies, in 572 (5.8%) appropriate for gestational age infants with congenital anomalies, and 809 (2.0%) appropriate for gestational age infants that experienced morbidity or mortality (with or without congenital anomalies). Genetic disorders were associated with an adjusted odds ratio (95% confidence interval) of 2.10 (1.89-2.33) of isolated small for gestational age and 12.84 (11.47-14.35) of small for gestational age accompanied by congenital anomalies. Genetic disorders were associated with an adjusted odds ratio of 2.24 (1.83-2.74) of morbidity or mortality., Conclusion: These findings suggest that genetic disorders are more prevalent in early preterm small for gestational age infants, particularly those with congenital anomalies. These findings also suggest that genetic disorders are associated with increased morbidity and mortality. These associations were primarily driven by trisomies 13, 18, and 21. Genetic diagnoses in this cohort were made through routine clinical care, principally via karyotype, chromosomal microarray, and single gene testing. These findings support evolving clinical guidelines for genetic testing of small for gestational age infants. Our study is limited due to the lack of prospective, genome-wide testing., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Childhood interstitial lung disease (chILD) secondary to pulmonary surfactant deficiency is a devastating chronic lung disease in children. Clinical presentation includes mild to severe respiratory failure and fibrosis. There is no specific treatment, except lung transplantation, which is hampered by a severe shortage of donor organs, especially for young patients. Repair of lungs with chILD represents a longstanding therapeutic challenge but cell therapy is a promising strategy. As surfactant is produced by alveolar type II epithelial (ATII) cells, engraftment with normal or gene-corrected ATII cells might provide an avenue to cure. Here we used a chILD disease-like model, Sftpc - / - mice, to provide proof-of-principle for this approach. Sftpc - / - mice developed chronic interstitial lung disease with age and were hypersensitive to bleomycin. We could engraft wild-type ATII cells after low dose bleomycin conditioning. Transplanted ATII cells produced mature SPC and attenuated bleomycin-induced lung injury up to two months post-transplant. This study demonstrates that partial replacement of mutant ATII cells can promote lung repair in a mouse model of chILD-like disease., Competing Interests: The authors have declared that no conflict of interest exists

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting over 300,000 people worldwide. It is characterized by the progressive decline of the nervous system that leads to the weakening of muscles which impacts physical function. Approximately, 15% of individuals diagnosed with ALS have a known genetic variant that contributes to their disease. As therapies that slow or prevent symptoms continue to develop, such as antisense oligonucleotides, it is important to discover novel genes that could be targets for treatment. Additionally, as cohorts continue to grow, performing analyses in ALS subtypes, such as primary lateral sclerosis (PLS), becomes possible due to an increase in power. These analyses could highlight novel pathways in disease manifestation., Methods: Building on our previous discoveries using rare variant association analyses, we conducted rare variant burden testing on a substantially larger multi-ethnic cohort of 6,970 ALS patients, 166 PLS patients, and 22,524 controls. We used intolerant domain percentiles based on sub-region Residual Variation Intolerance Score (subRVIS) that have been described previously in conjunction with gene based collapsing approaches to conduct burden testing to identify genes that associate with ALS and PLS., Results: A gene based collapsing model showed significant associations with SOD1, TARDBP, and TBK1 (OR = 19.18, p = 3.67 × 10 -39 ; OR = 4.73, p = 2 × 10 -10 ; OR = 2.3, p = 7.49 × 10 -9 , respectively). These genes have been previously associated with ALS. Additionally, a significant novel control enriched gene, ALKBH3 (p = 4.88 × 10 -7 ), was protective for ALS in this model. An intolerant domain-based collapsing model showed a significant improvement in identifying regions in TARDBP that associated with ALS (OR = 10.08, p = 3.62 × 10 -16 ). Our PLS protein truncating variant collapsing analysis demonstrated significant case enrichment in ANTXR2 (p = 8.38 × 10 -6 )., Conclusions: In a large multi-ethnic cohort of 6,970 ALS patients, collapsing analyses validated known ALS genes and identified a novel potentially protective gene, ALKBH3. A first-ever analysis in 166 patients with PLS found a candidate association with loss-of-function mutations in ANTXR2., (© 2024. The Author(s).)

Accurate estimation of population allele frequency (AF) is crucial for gene discovery and genetic diagnostics. However, determining AF for frameshift-inducing small insertions and deletions (indels) faces challenges due to discrepancies in mapping and variant calling methods. Here, we propose an innovative approach to assess indel AF. We developed CRAFTS-indels (Calculating Regional Allele Frequency Targeting Small indels), an algorithm that combines AF of distinct indels within a given region and provides "regional AF" (rAF). We tested and validated CRAFTS-indels using three independent datasets: gnomAD v2 (n=125,748 samples), an internal dataset (IGM; n=39,367), and the UK BioBank (UKBB; n=469,835). By comparing rAF against standard AF, we identified rare indels with rAF exceeding standard AF (sAF≤10 -4 and rAF>10 -4 ) as "rAF-hi" indels. Notably, a high percentage of rare indels were "rAF-hi", with a higher proportion in gnomAD v2 (11-20%) and IGM (11-22%) compared to the UKBB (5-9% depending on the CRAFTS-indels' parameters). Analysis of the overlap of regions based on their rAF with low complexity regions and with ClinVar classification supported the pertinence of rAF. Using the internal dataset, we illustrated the utility of CRAFTS-indel in the analysis of de novo variants and the potential negative impact of rAF-hi indels in gene discovery. In summary, annotation of indels with cohort specific rAF can be used to handle some of the limitations of current annotation pipelines and facilitate detection of novel gene disease associations. CRAFTS-indels offers a user-friendly approach to providing rAF annotation. It can be integrated into public databases such as gnomAD, UKBB and used by ClinVar to revise indel classifications., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: Congenital lymphatic anomalies (LAs) arise due to defects in lymphatic development and often present in utero as pleural effusion, chylothorax, nuchal and soft tissue edema, ascites, or hydrops. Many LAs are caused by single nucleotide variants, which are not detected on routine prenatal testing., Methods: Demographic data were compared between two subcohorts, those with clinically significant fetal edema (CSFE) and isolated fetal edema. A targeted variant analysis of LA genes was performed using American College of Medical Genetics criteria on whole exome sequencing (WES) data generated for 71 fetal edema cases who remained undiagnosed after standard workup., Results: CSFE cases had poor outcomes, including preterm delivery, demise, and maternal preeclampsia. Pathogenic and likely pathogenic variants were identified in 7% (5/71) of cases, including variants in RASopathy genes, RASA1, SOS1, PTPN11, and a novel PIEZO1 variant. Variants of uncertain significance (VOUS) were identified in 45% (32/71) of cases. In CSFEs, VOUS were found in CELSR1, EPHB4, TIE1, PIEZO1, ITGA9, RASopathy genes, SOS1, SOS2, and RAF1., Conclusions: WES identified pathogenic and likely pathogenic variants and VOUS in LA genes in 51% of fetal edema cases, supporting WES and expanded hydrops panels in cases of idiopathic fetal hydrops and fluid collections., (© 2023 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Childhood-onset schizophrenia (COS) is a rare form of schizophrenia with an onset prior to 13 years of age. Although genetic factors play a role in COS etiology, only a few causal variants have been reported to date. This study presents a diagnostic exome sequencing (ES) in 37 Israeli Jewish families with a proband diagnosed with COS. By implementing a trio/duo ES approach and applying a well-established diagnostic pipeline, we detected clinically significant variants in 7 probands (19 %). These single nucleotide variants and indels were mostly inherited. The implicated genes were ANKRD11, GRIA2, CHD2, CLCN3, CLTC, IGF1R and MICU1. In a secondary analysis that compared COS patients to 4721 healthy controls, we observed that patients had a significant enrichment of rare loss of function (LoF) variants in LoF intolerant genes associated with developmental diseases. Taken together, ES could be considered as a valuable tool in the genetic workup for COS patients., Competing Interests: Declaration of competing interest D.B.G. reports equity holdings in precision medicine companies (Q State – Pairnomix, Praxis Therapeutics, Apostle Inc., Actio Biosciences) and consultancy payments from Gilead Sciences, AstraZeneca, and GoldFinch Bio. A.A. is currently a full-time employee at the Regeneron Genetics Center and receives salary and stock options as compensation. All other authors have no conflict of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Importance: Diagnostic genetic testing can lead to changes in management in the pediatric intensive care unit. Genetic risk in children with critical illness but nondiagnostic exome sequencing (ES) has not been explored., Objective: To assess the association between loss-of-function (LOF) variants and pediatric critical illness., Design, Setting, and Participants: This genetic association study examined ES first screened for causative variants among 267 children at the Morgan Stanley Children's Hospital of NewYork-Presbyterian, of whom 22 were otherwise healthy with viral respiratory failure; 18 deceased children with bronchiolitis from the Office of the Chief Medical Examiner of New York City, of whom 14 were previously healthy; and 9990 controls from the Institute for Genomic Medicine at Columbia University Irving Medical Center. The ES data were generated between January 1, 2015, and December 31, 2020, and analyzed between January 1, 2017, and September 2, 2022., Exposure: Critical illness., Main Outcomes and Measures: Odds ratios and P values for genes and gene-sets enriched for rare LOF variants and the loss-of-function observed/expected upper bound fraction (LOEUF) score at which cases have a significant enrichment., Results: This study included 285 children with critical illness (median [range] age, 4.1 [0-18.9] years; 148 [52%] male) and 9990 controls. A total of 228 children (80%) did not receive a genetic diagnosis. After quality control (QC), 231 children harbored excess rare LOF variants in genes with a LOEUF score of 0.680 or less (intolerant genes) (P = 1.0 × 10-5). After QC, 176 children without a diagnosis harbored excess ultrarare LOF variants in intolerant genes but only in those without a known disease association (odds ratio, 1.8; 95% CI, 1.3-2.5). After QC, 25 children with viral respiratory failure harbored excess ultrarare LOF variants in intolerant genes but only in those without a known disease association (odds ratio, 2.8; 95% CI, 1.1-6.6). A total of 114 undiagnosed children were enriched for de novo LOF variants in genes without a known disease association (observed, 14; expected, 6.8; enrichment, 2.05)., Conclusions and Relevance: In this genetic association study, excess LOF variants were observed among critically ill children despite nondiagnostic ES. Variants lay in genes without a known disease association, suggesting future investigation may connect phenotypes to causative genes.

Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: We aimed to identify genes associated with genetic generalized epilepsy (GGE) by combining large cohorts enriched with individuals with a positive family history. Secondarily, we set out to compare the association of genes independently with familial and sporadic GGE., Methods: We performed a case-control whole exome sequencing study in unrelated individuals of European descent diagnosed with GGE (previously recruited and sequenced through multiple international collaborations) and ancestry-matched controls. The association of ultra-rare variants (URVs; in 18 834 protein-coding genes) with epilepsy was examined in 1928 individuals with GGE (vs. 8578 controls), then separately in 945 individuals with familial GGE (vs. 8626 controls), and finally in 1005 individuals with sporadic GGE (vs. 8621 controls). We additionally examined the association of URVs with familial and sporadic GGE in two gene sets important for inhibitory signaling (19 genes encoding γ-aminobutyric acid type A [GABA A ] receptors, 113 genes representing the GABAergic pathway)., Results: GABRG2 was associated with GGE (p = 1.8 × 10 -5 ), approaching study-wide significance in familial GGE (p = 3.0 × 10 -6 ), whereas no gene approached a significant association with sporadic GGE. Deleterious URVs in the most intolerant subgenic regions in genes encoding GABA A receptors were associated with familial GGE (odds ratio [OR] = 3.9, 95% confidence interval [CI] = 1.9-7.8, false discovery rate [FDR]-adjusted p = .0024), whereas their association with sporadic GGE had marginally lower odds (OR = 3.1, 95% CI = 1.3-6.7, FDR-adjusted p = .022). URVs in GABAergic pathway genes were associated with familial GGE (OR = 1.8, 95% CI = 1.3-2.5, FDR-adjusted p = .0024) but not with sporadic GGE (OR = 1.3, 95% CI = .9-1.9, FDR-adjusted p = .19)., Significance: URVs in GABRG2 are likely an important risk factor for familial GGE. The association of gene sets of GABAergic signaling with familial GGE is more prominent than with sporadic GGE., (© 2022 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Extreme phenotype sequencing has led to the identification of high-impact rare genetic variants for many complex disorders but has not been applied to studies of severe schizophrenia. We sequenced 112 individuals with severe, extremely treatment-resistant schizophrenia, 218 individuals with typical schizophrenia, and 4,929 controls. We compared the burden of rare, damaging missense and loss-of-function variants between severe, extremely treatment-resistant schizophrenia, typical schizophrenia, and controls across mutation intolerant genes. Individuals with severe, extremely treatment-resistant schizophrenia had a high burden of rare loss-of-function (odds ratio, 1.91; 95% CI, 1.39 to 2.63; P = 7.8 × 10 -5 ) and damaging missense variants in intolerant genes (odds ratio, 2.90; 95% CI, 2.02 to 4.15; P = 3.2 × 10 -9 ). A total of 48.2% of individuals with severe, extremely treatment-resistant schizophrenia carried at least one rare, damaging missense or loss-of-function variant in intolerant genes compared to 29.8% of typical schizophrenia individuals (odds ratio, 2.18; 95% CI, 1.33 to 3.60; P = 1.6 × 10 -3 ) and 25.4% of controls (odds ratio, 2.74; 95% CI, 1.85 to 4.06; P = 2.9 × 10 -7 ). Restricting to genes previously associated with schizophrenia risk strengthened the enrichment with 8.9% of individuals with severe, extremely treatment-resistant schizophrenia carrying a damaging missense or loss-of-function variant compared to 2.3% of typical schizophrenia (odds ratio, 5.48; 95% CI, 1.52 to 19.74; P = 0.02) and 1.6% of controls (odds ratio, 5.82; 95% CI, 3.00 to 11.28; P = 2.6 × 10 -8 ). These results demonstrate the power of extreme phenotype case selection in psychiatric genetics and an approach to augment schizophrenia gene discovery efforts., Competing Interests: Competing interest statement: D.B.G. is a founder of and holds equity in Q State Biosciences and Praxis Therapeutics; holds equity in Apostle Inc., and serves as a consultant to AstraZeneca, Gilead Sciences, GoldFinch Bio and Gossamer Bio. J.A.L. does not accept any personal financial remuneration for consulting, speaking, or research activities from any pharmaceutical, biotechnology, or medical device companies. He receives funding and medication supplies for investigator-initiated research from Denovo, Taisho, and Cerevel, and company sponsored phase II, III, and IV studies from Alkermes, Sunovion, and Boehringer Ingelheim, which does not contribute to his compensation. He is a consultant or advisory board member of Intracellular Therapies, Takeda, Karuna, Pear Therapeutics, Systems-1, and Psychogenics for which he receives no remuneration. He is a paid consultant for Signant Health, a clinical research technology and services organization, and holds a patent from Repligen that yields no royalties. R.S.D. is a paid consultant for AstraZeneca. X.W. is a cofounder and stock owner at Waypoint Bio. The other authors declare no competing interests.

Introduction: Social determinants of health (SDOH) account for 80% of modifiable factors in a population's health. Addressing SDOH in a healthcare setting can improve care, patient experience, health outcomes, and decrease cost. Therefore, screening for SODH in the pediatric setting has become an essential and evidence-based component of pediatric preventative care. Multiple barriers exist for its implementation, particularly for trainees., Methods: Using resident-driven quality improvement (QI) methodology, we aimed to increase SDOH screening to >90% for 9 individual questions at newborn and 1-year well visits and completely screen for all 9 questions at more than 40% of visits. Parents were provided with a paper screening form upon arrival to be completed before visits. We performed tests of change to improve distribution, documentation, and quality of interventions., Results: The primary outcome of complete screening for all 9 questions increased from 24% to 43% at newborn visits and 28% to 83% at 1-year visits. Screenings that identified at least 1 need increased from 8% to 19%, with provider response to an identified need increasing from 20% to 40%. These metrics were the secondary outcome measures., Conclusions: The use of parent completed paper screening forms improved SDOH screening, documentation, and interventions by residents and faculty., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

The thalamus plays diverse roles in cortical-subcortical brain activity patterns. Recent work suggests that focal temporal lobe seizures depress subcortical arousal systems and convert cortical activity into a pattern resembling slow-wave sleep. The potential simultaneous and paradoxical role of the thalamus in both limbic seizure propagation, and in sleep-like cortical rhythms has not been investigated. We recorded neuronal activity from the central lateral (CL), anterior (ANT), and ventral posteromedial (VPM) nuclei of the thalamus in an established female rat model of focal limbic seizures. We found that population firing of neurons in CL decreased during seizures while the cortex exhibited slow waves. In contrast, ANT showed a trend toward increased neuronal firing compatible with polyspike seizure discharges seen in the hippocampus. Meanwhile, VPM exhibited a remarkable increase in sleep spindles during focal seizures. Single-unit juxtacellular recordings from CL demonstrated reduced overall firing rates, but a switch in firing pattern from single spikes to burst firing during seizures. These findings suggest that different thalamic nuclei play very different roles in focal limbic seizures. While limbic nuclei, such as ANT, appear to participate directly in seizure propagation, arousal nuclei, such as CL, may contribute to depressed cortical function, whereas sleep spindles in relay nuclei, such as VPM, may interrupt thalamocortical information flow. These combined effects could be critical for controlling both seizure severity and impairment of consciousness. Further understanding of differential effects of seizures on different thalamocortical networks may lead to improved treatments directly targeting these modes of impaired function. SIGNIFICANCE STATEMENT Temporal lobe epilepsy has a major negative impact on quality of life. Previous work suggests that the thalamus plays a critical role in thalamocortical network modulation and subcortical arousal maintenance, but its precise seizure-associated functions are not known. We recorded neuronal activity in three different thalamic regions and found divergent activity patterns, which may respectively participate in seizure propagation, impaired level of conscious arousal, and altered relay of information to the cortex during focal limbic seizures. These very different activity patterns within the thalamus may help explain why focal temporal lobe seizures often disrupt widespread network function, and can help guide future treatments aimed at restoring normal thalamocortical network activity and cognition., (Copyright © 2017 the authors 0270-6474/17/3711441-14$15.00/0.)

Impaired breathing, cardiac function, and arousal during and after seizures are important causes of morbidity and mortality. Previous work suggests that these changes are associated with depressed brainstem function in the ictal and post-ictal periods. Lower brainstem serotonergic systems are postulated to play an important role in cardiorespiratory changes during and after seizures, whereas upper brainstem serotonergic and other systems regulate arousal. However, direct demonstration of seizure-associated neuronal activity changes in brainstem serotonergic regions has been lacking. Here, we performed multiunit and single-unit recordings from medullary raphe and midbrain dorsal raphe nuclei in an established rat seizure model while measuring changes in breathing rate and depth as well as heart rate. Serotonergic neurons were identified by immunohistochemistry. Respiratory rate, tidal volume, and minute ventilation were all significantly decreased during and after seizures in this model. We found that population firing of neurons in the medullary and midbrain raphe on multiunit recordings was significantly decreased during the ictal and post-ictal periods. Single-unit recordings from identified serotonergic neurons in the medullary raphe revealed highly consistently decreased firing during and after seizures. In contrast, firing of midbrain raphe serotonergic neurons was more variable, with a mixture of increases and decreases. The markedly suppressed firing of medullary serotonergic neurons supports their possible role in simultaneously impaired cardiorespiratory function in seizures. Decreased arousal likely arises from depressed population activity of several neuronal pools in the upper brainstem and forebrain. These findings have important implications for preventing morbidity and mortality in people living with epilepsy., Significance Statement: Seizures often cause impaired breathing, cardiac dysfunction, and loss of consciousness. The brainstem and, specifically, brainstem serotonin neurons are thought to play an important role in controlling breathing, cardiac function, and arousal. We used an established rat seizure model to study the overall neuronal activity in the brainstem as well as firing of specific serotonin neurons while measuring cardiorespiratory function. Our results demonstrated overall decreases in brainstem neuronal activity and marked downregulation of lower brainstem serotonin neuronal firing in association with decreased breathing and heart rate during and after seizures. These findings point the way toward new treatments to augment brainstem function and serotonin, aiming to prevent seizure complications and reduce morbidity and mortality in people living with epilepsy., (Copyright © 2016 the authors 0270-6474/16/362712-12$15.00/0.)

Focal temporal lobe seizures often cause impaired cortical function and loss of consciousness. Recent work suggests that the mechanism for depressed cortical function during focal seizures may depend on decreased subcortical cholinergic arousal, which leads to a sleep-like state of cortical slow-wave activity. To test this hypothesis, we sought to directly activate subcortical cholinergic neurons during focal limbic seizures to determine the effects on cortical function. Here we used an optogenetic approach to selectively stimulate cholinergic brainstem neurons in the pedunculopontine tegmental nucleus during focal limbic seizures induced in a lightly anesthetized rat model. We found an increase in cortical gamma activity and a decrease in delta activity in response to cholinergic stimulation. These findings support the mechanistic role of reduced subcortical cholinergic arousal in causing cortical dysfunction during seizures. Through further work, electrical or optogenetic stimulation of subcortical arousal networks may ultimately lead to new treatments aimed at preventing cortical dysfunction during seizures., (Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.)

Absence seizures are transient episodes of impaired consciousness accompanied by 3-4 Hz spike-wave discharge on electroencephalography (EEG). Human functional magnetic resonance imaging (fMRI) studies have demonstrated widespread cortical decreases in the blood oxygen-level dependent (BOLD) signal that may play an important role in the pathophysiology of these seizures. Animal models could provide an opportunity to investigate the fundamental mechanisms of these changes, however they have so far failed to consistently replicate the cortical fMRI decreases observed in human patients. This may be due to important differences between human seizures and animal models, including a lack of cortical development in rodents or differences in the frequencies of rodent (7-8 Hz) and human (3-4 Hz) spike-wave discharges. To examine the possible contributions of these differences, we developed a ferret model that exhibits 3-4 Hz spike-wave seizures in the presence of a sulcated cortex. Measurements of BOLD fMRI and simultaneous EEG demonstrated cortical fMRI increases during and following spike-wave seizures in ferrets. However unlike human patients, significant fMRI decreases were not observed. The lack of fMRI decreases was consistent across seizures of different durations, discharge frequencies, and anesthetic regimes, and using fMRI analysis models similar to human patients. In contrast, generalized tonic-clonic seizures under the same conditions elicited sustained postictal fMRI decreases, verifying that the lack of fMRI decreases with spike-wave was not due to technical factors. These findings demonstrate that 3-4 Hz spike-wave discharge in a sulcated animal model does not necessarily produce fMRI decreases, leaving the mechanism for this phenomenon open for further investigation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Impaired consciousness in temporal lobe seizures has a major negative impact on quality of life. The prevailing view holds that this disorder impairs consciousness by seizure spread to the bilateral temporal lobes. We propose instead that seizures invade subcortical regions and depress arousal, causing impairment through decreases rather than through increases in activity. Using functional magnetic resonance imaging in a rodent model, we found increased activity in regions known to depress cortical function, including lateral septum and anterior hypothalamus. Importantly, we found suppression of intralaminar thalamic and brainstem arousal systems and suppression of the cortex. At a cellular level, we found reduced firing of identified cholinergic neurons in the brainstem pedunculopontine tegmental nucleus and basal forebrain. Finally, we used enzyme-based amperometry to demonstrate reduced cholinergic neurotransmission in both cortex and thalamus. Decreased subcortical arousal is a critical mechanism for loss of consciousness in focal temporal lobe seizures., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Understanding the neural mechanisms that support human consciousness is an important frontier in neuroscience and medicine. We previously developed a rodent model of temporal lobe seizures that recapitulates the human electroencephalography (EEG) signature of ictal and postictal neocortical slow waves associated with behavioral impairments in level of consciousness. The mechanism of slow-wave production in epilepsy may involve suppression of the subcortical arousal systems including the brainstem and intralaminar thalamic nuclei. We hypothesized that intralaminar thalamic stimulation may lead to electrophysiologic and functional rescue from postictal slow waves and behavioral arrest., Methods: We electrically stimulated the central lateral thalamic nucleus (a member of the intralaminar nuclei) under anesthesia and after electrically induced hippocampal seizures in anesthetized and in awake-behaving animal model preparations., Results: We demonstrated a proof-of-principle restoration of electrophysiologic and behavioral measures of consciousness by stimulating the intralaminar thalamic nuclei after seizures. We measured decreased cortical slow waves and increased desynchronization and multiunit activity in the cortex with thalamic stimulation following seizures. Functionally, thalamic stimulation produced resumption of exploratory behaviors in the postictal state., Significance: Targeting of nodes in the neural circuitry of consciousness has important medical implications. Impaired consciousness with epilepsy has dangerous consequences including decreased school/work performance, social stigmatization, and impaired airway protection. These data suggest a novel therapeutic approach for restoring consciousness after seizures. If paired with responsive neurostimulation, this may allow rapid implementation to improve level of consciousness in patients with epilepsy., (Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.)

Background: Cortical networks undergo large-scale switching between states of increased or decreased activity in normal sleep and cognition as well as in pathological conditions such as epilepsy. We previously found that focal hippocampal seizures in rats induce increased neuronal firing and cerebral blood flow in subcortical structures including the lateral septal area, along with frontal cortical slow oscillations resembling slow wave sleep. In addition, stimulation of the lateral septum in the absence of a seizure resulted in cortical deactivation with slow oscillations., Hypothesis: We hypothesized that lateral septal activation might cause neocortical deactivation indirectly, possibly through impaired subcortical arousal. But how does subcortical stimulation cause slow wave activity in frontal cortex? How do arousal neurotransmitter levels (e.g. acetylcholine) change in cortex during the excitation of inhibitory projection nuclei?, Methods and Results: In the current study, we used simultaneous electrophysiology and enzyme-based amperometry in a rat model, and found a decrease in choline, along with slow wave activity in orbital frontal cortex during lateral septal stimulation in the absence of seizures. In contrast, the choline signal and local field potential in frontal cortex had no significant changes when stimulating the hippocampus, but showed increased choline and decreased slow wave activity with an arousal stimulus produced by toe pinch., Conclusions: These findings indicate that the activation of subcortical inhibitory structures (such as lateral septum) can depress subcortical cholinergic arousal. This mechanism may play an important role in large-scale transitions of cortical activity in focal seizures, as well as in normal cortical function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Functional magnetic resonance imaging (fMRI)-based resting functional connectivity is well suited for measuring slow correlated activity throughout brain networks. Epilepsy involves chronic changes in normal brain networks, and recent work demonstrated enhanced resting fMRI connectivity between the hemispheres in childhood absence epilepsy. An animal model of this phenomenon would be valuable for investigating fundamental mechanisms and testing therapeutic interventions., Methods: We used fMRI-based resting functional connectivity for studying brain networks involved in absence epilepsy. Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) exhibit spontaneous episodes of staring and unresponsiveness accompanied by spike-wave discharges (SWDs) resembling human absence seizures in behavior and electroencephalography (EEG). Simultaneous EEG-fMRI data in epileptic WAG/Rij rats in comparison to nonepileptic Wistar controls were acquired at 9.4 T. Regions showing cortical fMRI increases during SWDs were used to define reference regions for connectivity analysis to investigate whether chronic seizure activity is associated with changes in network resting functional connectivity., Key Findings: We observed high degrees of cortical-cortical correlations in all WAG/Rij rats at rest (when no SWDs were present), but not in nonepileptic controls. Strongest connectivity was seen between regions most intensely involved in seizures, mainly in the bilateral somatosensory and adjacent cortices. Group statistics revealed that resting interhemispheric cortical-cortical correlations were significantly higher in WAG/Rij rats compared to nonepileptic controls., Significance: These findings suggest that activity-dependent plasticity may lead to long-term changes in epileptic networks even at rest. The results show a marked difference between the epileptic and nonepileptic animals in cortical-cortical connectivity, indicating that this may be a useful interictal biomarker associated with the epileptic state., (Wiley Periodicals, Inc. © 2013 International League Against Epilepsy.)

Impaired consciousness in epilepsy has a major negative impact on quality of life. Prior work suggests that complex partial seizures (CPS) and generalized tonic-clonic seizures (GTCS), which both cause loss of consciousness, affect similar frontoparietal networks. Milder involvement in CPS than in GTCS may spare some simple behavioral responses, resembling the minimally conscious state. However, this difference in responses has not been rigorously tested previously. During video-electroencephalography (EEG) monitoring, we administered a standardized prospective testing battery including responses to questions and commands, as well as tests for reaching/grasping a ball and visual tracking in 27 CPS (in 14 patients) and 7 GTCS (in six patients). Behavioral results were analyzed in the ictal and postictal periods based on video review. During both CPS and GTCS, patients were unable to respond to questions or commands. However, during CPS, patients often retained minimally conscious ball grasping and visual tracking responses. Patients were able to successfully grasp a ball in 60% or to visually track in 58% of CPS, and could carry out both activities in 52% of CPS. In contrast, during GTCS, preserved ball grasp (10%), visual tracking (11%), or both (7%), were all significantly less than in CPS. Postictal ball grasping and visual tracking were also somewhat better following CPS than GTCS. These findings suggest that impaired consciousness in CPS is more similar to minimally conscious state than to coma. Further work may elucidate the specific brain networks underlying relatively spared functions in CPS, ultimately leading to improved treatments aimed at preventing impaired consciousness., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

The relationship between neuronal activity and hemodynamic changes plays a central role in functional neuroimaging. Under normal conditions and in neurological disorders such as epilepsy, it is commonly assumed that increased functional magnetic resonance imaging (fMRI) signals reflect increased neuronal activity and that fMRI decreases represent neuronal activity decreases. Recent work suggests that these assumptions usually hold true in the cerebral cortex. However, less is known about the basis of fMRI signals from subcortical structures such as the thalamus and basal ganglia. We used WAG/Rij rats (Wistar albino Glaxo rats of Rijswijk), an established animal model of human absence epilepsy, to perform fMRI studies with blood oxygen level-dependent and cerebral blood volume (CBV) contrasts at 9.4 tesla, as well as laser Doppler cerebral blood flow (CBF), local field potential (LFP), and multiunit activity (MUA) recordings. We found that, during spike-wave discharges, the somatosensory cortex and thalamus showed increased fMRI, CBV, CBF, LFP, and MUA signals. However, the caudate-putamen showed fMRI, CBV, and CBF decreases despite increases in LFP and MUA signals. Similarly, during normal whisker stimulation, the cortex and thalamus showed increases in CBF and MUA, whereas the caudate-putamen showed decreased CBF with increased MUA. These findings suggest that neuroimaging-related signals and electrophysiology tend to agree in the cortex and thalamus but disagree in the caudate-putamen. These opposite changes in vascular and electrical activity indicate that caution should be applied when interpreting fMRI signals in both health and disease from the caudate-putamen, as well as possibly from other subcortical structures.

Impaired consciousness requires altered cortical function. This can occur either directly from disorders that impair widespread bilateral regions of the cortex or indirectly through effects on subcortical arousal systems. It has therefore long been puzzling why focal temporal lobe seizures so often impair consciousness. Early work suggested that altered consciousness may occur with bilateral or dominant temporal lobe seizure involvement. However, other bilateral temporal lobe disorders do not impair consciousness. More recent work supports a 'network inhibition hypothesis' in which temporal lobe seizures disrupt brainstem-diencephalic arousal systems, leading indirectly to depressed cortical function and impaired consciousness. Indeed, prior studies show subcortical involvement in temporal lobe seizures and bilateral frontoparietal slow wave activity on intracranial electroencephalography. However, the relationships between frontoparietal slow waves and impaired consciousness and between cortical slowing and fast seizure activity have not been directly investigated. We analysed intracranial electroencephalography recordings during 63 partial seizures in 26 patients with surgically confirmed mesial temporal lobe epilepsy. Behavioural responsiveness was determined based on blinded review of video during seizures and classified as impaired (complex-partial seizures) or unimpaired (simple-partial seizures). We observed significantly increased delta-range 1-2 Hz slow wave activity in the bilateral frontal and parietal neocortices during complex-partial compared with simple-partial seizures. In addition, we confirmed prior work suggesting that propagation of unilateral mesial temporal fast seizure activity to the bilateral temporal lobes was significantly greater in complex-partial than in simple-partial seizures. Interestingly, we found that the signal power of frontoparietal slow wave activity was significantly correlated with the temporal lobe fast seizure activity in each hemisphere. Finally, we observed that complex-partial seizures were somewhat more common with onset in the language-dominant temporal lobe. These findings provide direct evidence for cortical dysfunction in the form of bilateral frontoparietal slow waves associated with impaired consciousness in temporal lobe seizures. We hypothesize that bilateral temporal lobe seizures may exert a powerful inhibitory effect on subcortical arousal systems. Further investigations will be needed to fully determine the role of cortical-subcortical networks in ictal neocortical dysfunction and may reveal treatments to prevent this important negative consequence of temporal lobe epilepsy.

Purpose: Absence seizures cause transient impairment of consciousness. Typical absence seizures occur in children, and are accompanied by 3-4-Hz spike-wave discharges (SWDs) on electroencephalography (EEG). Prior EEG-functional magnetic resonance imaging (fMRI) studies of SWDs have shown a network of cortical and subcortical changes during these electrical events. However, fMRI during typical childhood absence seizures with confirmed impaired consciousness has not been previously investigated., Methods: We performed EEG-fMRI with simultaneous behavioral testing in 37 children with typical childhood absence epilepsy (CAE). Attentional vigilance was evaluated by a continuous performance task (CPT), and simpler motor performance was evaluated by a repetitive tapping task (RTT)., Results: SWD episodes were obtained during fMRI scanning from 9 patients among the 37 studied. fMRI signal increases during SWDs were observed in the thalamus, frontal cortex, primary visual, auditory, somatosensory, and motor cortex, and fMRI decreases were seen in the lateral and medial parietal cortex, cingulate gyrus, and basal ganglia. Omission error rate (missed targets) with SWDs during fMRI was 81% on CPT and 39% on RTT. For those seizure epochs during which CPT performance was impaired, fMRI changes were seen in cortical and subcortical structures typically involved in SWDs, whereas minimal changes were observed for the few epochs during which performance was spared., Discussion: These findings suggest that typical absence seizures involve a network of cortical-subcortical areas necessary for normal attention and primary information processing. Identification of this network may improve understanding of cognitive impairments in CAE, and may help guide development of new therapies for this disorder., (Wiley Periodicals, Inc. © 2010 International League Against Epilepsy.)

Patients with epilepsy are at risk of traffic accidents when they have seizures while driving. However, driving is an essential part of normal daily life in many communities, and depriving patients of driving privileges can have profound consequences for their economic and social well-being. In the current study, we collected ictal performance data from a driving simulator and two other video games in patients undergoing continuous video/EEG monitoring. We captured 22 seizures in 13 patients and found that driving impairment during seizures differed in terms of both magnitude and character, depending on the seizure type. Our study documents the feasibility of a prospective study of driving and other behaviors during seizures through the use of computer-based tasks. This methodology may be applied to further describe differential driving impairment in specific types of seizures and to gain data on anatomical networks disrupted in seizures that impair consciousness and driving safety., (Copyright 2010 Elsevier Inc. All rights reserved.)

Generalized tonic-clonic seizures cause widespread physiological changes throughout the cerebral cortex and subcortical structures in the brain. Using combined blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) at 9.4 T and electroencephalography (EEG), these changes can be characterized with high spatiotemporal resolution. We studied BOLD changes in anesthetized Wistar rats during bicuculline-induced tonic-clonic seizures. Bicuculline, a GABA(A) receptor antagonist, was injected systemically and seizure activity was observed on EEG as high-amplitude, high-frequency polyspike discharges followed by clonic paroxysmal activity of lower frequency, with mean electrographic seizure duration of 349 s. Our aim was to characterize the spatial localization, direction, and timing of BOLD signal changes during the pre-ictal, ictal and post-ictal periods. Group analysis was performed across seizures using paired t-maps of BOLD signal superimposed on high-resolution anatomical images. Regional analysis was then performed using volumes of interest to quantify BOLD timecourses. In the pre-ictal period we found focal BOLD increases in specific areas of somatosensory cortex (S1, S2) and thalamus several seconds before seizure onset. During seizures we observed BOLD increases in cortex, brainstem and thalamus and BOLD decreases in the hippocampus. The largest ictal BOLD increases remained in the focal regions of somatosensory cortex showing pre-ictal increases. During the post-ictal period we observed widespread BOLD decreases. These findings support a model in which "generalized" tonic-clonic seizures begin with focal changes before electrographic seizure onset, which progress to non-uniform changes during seizures, possibly shedding light on the etiology and pathophysiology of similar seizures in humans., (Copyright 2010 Elsevier Inc. All rights reserved.)

Partial seizures produce increased cerebral blood flow in the region of seizure onset. These regional cerebral blood flow increases can be detected by single photon emission computed tomography (ictal SPECT), providing a useful clinical tool for seizure localization. However, when partial seizures secondarily generalize, there are often questions of interpretation since propagation of seizures could produce ambiguous results. Ictal SPECT from secondarily generalized seizures has not been thoroughly investigated. We analysed ictal SPECT from 59 secondarily generalized tonic-clonic seizures obtained during epilepsy surgery evaluation in 53 patients. Ictal versus baseline interictal SPECT difference analysis was performed using ISAS (http://spect.yale.edu). SPECT injection times were classified based on video/EEG review as either pre-generalization, during generalization or in the immediate post-ictal period. We found that in the pre-generalization and generalization phases, ictal SPECT showed significantly more regions of cerebral blood flow increases than in partial seizures without secondary generalization. This made identification of a single unambiguous region of seizure onset impossible 50% of the time with ictal SPECT in secondarily generalized seizures. However, cerebral blood flow increases on ictal SPECT correctly identified the hemisphere (left versus right) of seizure onset in 84% of cases. In addition, when a single unambiguous region of cerebral blood flow increase was seen on ictal SPECT, this was the correct localization 80% of the time. In agreement with findings from partial seizures without secondary generalization, cerebral blood flow increases in the post-ictal period and cerebral blood flow decreases during or following seizures were not useful for localizing seizure onset. Interestingly, however, cerebral blood flow hypoperfusion during the generalization phase (but not pre-generalization) was greater on the side opposite to seizure onset in 90% of patients. These findings suggest that, with appropriate cautious interpretation, ictal SPECT in secondarily generalized seizures can help localize the region of seizure onset.

Generalized tonic-clonic seizures are among the most dramatic physiological events in the nervous system. The brain regions involved during partial seizures with secondary generalization have not been thoroughly investigated in humans. We used single photon emission computed tomography (SPECT) to image cerebral blood flow (CBF) changes in 59 secondarily generalized seizures from 53 patients. Images were analysed using statistical parametric mapping to detect cortical and subcortical regions most commonly affected in three different time periods: (i) during the partial seizure phase prior to generalization; (ii) during the generalization period; and (iii) post-ictally. We found that in the pre-generalization period, there were focal CBF increases in the temporal lobe on group analysis, reflecting the most common region of partial seizure onset. During generalization, individual patients had focal CBF increases in variable regions of the cerebral cortex. Group analysis during generalization revealed that the most consistent increase occurred in the superior medial cerebellum, thalamus and basal ganglia. Post-ictally, there was a marked progressive CBF increase in the cerebellum which spread to involve the bilateral lateral cerebellar hemispheres, as well as CBF increases in the midbrain and basal ganglia. CBF decreases were seen in the fronto-parietal association cortex, precuneus and cingulate gyrus during and following seizures, similar to the 'default mode' regions reported previously to show decreased activity in seizures and in normal behavioural tasks. Analysis of patient behaviour during and following seizures showed impaired consciousness at the time of SPECT tracer injections. Correlation analysis across patients demonstrated that cerebellar CBF increases were related to increases in the upper brainstem and thalamus, and to decreases in the fronto-parietal association cortex. These results reveal a network of cortical and subcortical structures that are most consistently involved in secondarily generalized tonic-clonic seizures. Abnormal increased activity in subcortical structures (cerebellum, basal ganglia, brainstem and thalamus), along with decreased activity in the association cortex may be crucial for motor manifestations and for impaired consciousness in tonic-clonic seizures. Understanding the networks involved in generalized tonic-clonic seizures can provide insights into mechanisms of behavioural changes, and may elucidate targets for improved therapies.

Generalized spike-wave seizures are typically brief events associated with dynamic changes in brain physiology, metabolism, and behavior. Functional magnetic resonance imaging (fMRI) provides a relatively high spatiotemporal resolution method for imaging cortical-subcortical network activity during spike-wave seizures. Patients with spike-wave seizures often have episodes of staring and unresponsiveness which interfere with normal behavior. Results from human fMRI studies suggest that spike-wave seizures disrupt specific networks in the thalamus and frontoparietal association cortex which are critical for normal attentive consciousness. However, the neuronal activity underlying imaging changes seen during fMRI is not well understood, particularly in abnormal conditions such as seizures. Animal models have begun to provide important fundamental insights into the neuronal basis for fMRI changes during spike-wave activity. Work from these models including both fMRI and direct neuronal recordings suggest that, in humans, specific cortical-subcortical networks are involved in spike-wave, while other regions are spared. Regions showing fMRI increases demonstrate correlated increases in neuronal activity in animal models. The mechanisms of fMRI decreases in spike-wave will require further investigation. A better understanding of the specific brain regions involved in generating spike-wave seizures may help guide efforts to develop targeted therapies aimed at preventing or reversing abnormal excitability in these brain regions, ultimately leading to a cure for this disorder.

Blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) is widely used in neuroscience to study brain activity. However, BOLD fMRI does not measure neuronal activity directly but depends on cerebral blood flow (CBF), cerebral blood volume (CBV), and cerebral metabolic rate of oxygen (CMRO(2)) consumption. Using fMRI, CBV, CBF, neuronal recordings, and CMRO(2) modeling, we investigated how the signals are related during seizures in rats. We found that increases in hemodynamic, neuronal, and metabolic activity were associated with positive BOLD signals in the cortex, but with negative BOLD signals in hippocampus. Our data show that negative BOLD signals do not necessarily imply decreased neuronal activity or CBF, but can result from increased neuronal activity, depending on the interplay between hemodynamics and metabolism. Caution should be used in interpreting fMRI signals because the relationship between neuronal activity and BOLD signals may depend on brain region and state and can be different during normal and pathological conditions.