NOG gene and affected non-Boxer dogs without the deletion were performed.Superficial chronic corneal epithelial defects (SCCEDs) are spontaneous corneal defects in dogs that share many clinical and pathologic characteristics to recurrent corneal erosions (RCE) in humans. Boxer dogs are predisposed to SCCEDs, therefore a search for a genetic defect was performed to explain this susceptibility. DNA was extracted from blood collected from Boxer dogs with and without SCCEDs followed by whole genome sequencing (WGS). RNA sequencing of corneal tissue and immunostaining of corneal sections from affected SCCED Boxer dogs with a deletion in the NOG) (Chr 9:31453999) was identified by WGS and was significantly associated (P < 0.0001) with Boxer SCCEDs compared to unaffected non-Boxer dogs. NOG, BMP4, MMP13, and NCAM1 all had significant fold reductions in expression and SHH was significantly increased in Boxers with the NOG deletion as identified by RNA-Seq. Corneal IHC from NOG deletion dogs with SCCEDs had lower NOG and significantly higher scores of BMP2.A 30 base pair deletion at a splice site in Noggin . Dysregulation of LEPC may play a role in the pathogenesis of RCE.Many Boxer dogs with SCCED have a genetic defect in The online version contains supplementary material available at 10.1186/s12917-021-02955-1. Superficial chronic corneal epithelial defects (SCCEDs), also known as indolent corneal ulcers, are spontaneous corneal defects in dogs that share many clinical, pathophysiologic, and pathologic characteristics with recurrent corneal erosions (RCE) in humans –3. TheseTreatment of SCCEDs or RCEs remains challenging and therapies for these chronic, painful conditions have not changed substantially in the past several decades. Conservative treatments, such as epithelial debridement, topical antibiotics, and artificial tears are commonly used but rarely effective. Unresponsive patients may benefit from use of therapeutic bandage contact lenses, amnion membrane grafts, matrix metalloproteinase (MMP) inhibitors , and/or topical serum eye drops. Diamond burr debridement, anterior stromal puncture, alcohol delamination, or superficial keratectomy are used in resistant cases, especially when the patient has suspected epithelial basement membrane dystrophies –6, 10–15NOG), which was significantly associated (P < 0.0001) with Boxer dogs with SCCEDs compared to unaffected non-Boxer dogs. Furthermore, RNA sequencing of corneal samples and immunostaining of keratectomy specimens from SCCED Boxer dogs deficient in NOG revealed reduced expression of NOG and alteration of RNA expression of several factors in the BMP signaling pathway compared to non-Boxer SCCED dogs that were wild type (WT) for the NOG deletion. These results suggest that NOG deficiency and effect on the BMP signaling pathway may impact limbal cell progenitor cells and play a role in SCCEDs and RCEs.Boxer dogs are highly predisposed to development of SCCEDs, however, the underlying cause is unknown , 16–18. Use of animals in this study adhered to the Association for Research in Vision and Ophthalmology statement on the use of animals in ophthalmic and vision research. The animal use protocol was approved and monitored by the North Carolina State University Institutional Animal Care and Use Committee (IACUC) (Protocol # 18–164-0). The North Carolina State University Veterinary Hospital Board approved the protocol for evaluation of clinical patients in this study. Owners of dogs signed and provided informed consent for all sample collections and data collection. The study was carried out in compliance with the ARRIVE guidelines.Whole blood samples were collected from Boxer dogs either with, or having a documented history  of having SCCED, and Boxer dogs over 10 years of age . with no known history of a SCCED and no evidence of previous corneal disease as examined by an NC State University veterinary ophthalmologist. All blood was collected in EDTA tubes and DNA was extracted using the standard protocol of the DNeasy Blood and Tissue Kit .Samples from 8 SCCED Boxer dogs were submitted for library preparation and whole genome sequencing, using a 150 base pair (bp) paired-end read configuration on an Illumina HiSeq 4000 high-throughput sequencing system .http://broadinstitute.github.io/picard) and GATK 3.7 following best practices for base quality score recalibration and indel realignment  [Variant calling from next-generation sequencing data was performed using a standardized bioinformatics pipeline for all samples, as described previously . SequencMA, USA) , 24. Varhttps://cidd.discoveryspace.ca/).  and prioritized by their functional impact . They were manually curated for potential role in corneal disease and corneal wound healing, such as bone morphogenetic proteins (BMPs), transforming growth factor beta superfamily, noggin, etc. Missense variants were evaluated for genomic functional significance with Polyphen (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/) and Provean (http://provean.jcvi.org/index.php). In Silico Splice Site Analysis was performed with Human Slice Site Finder (http://www.umd.be/HSF/) to predict the potential impact of splice site changes.Variants (heterozygous or homozygous) present in at least 7/8 affected Boxers were identified. The resulting variants were then filtered against a previously established database of variants from 84 non-Boxer dogs of 17 different dog breeds without a known risk for development of SCCED  and were pursued with Sanger Sequencing in the additional affected (62) Boxer dogs and apparently unaffected (25) Boxer dogs and tested for allelic association with SCCED using a Fisher’s exact test. A Variants of strongest interest were subsequently filtered against a larger established canine database of 391 non-Boxer dogs of 53 different breeds to assess variant prevalence in the canine population.NOG deletion and three affected non-Boxer dogs without the NOG deletion (Boston Terrier (2), Pomeranian) as has been described previously [) as well as by quantitative PCR . The sequencing libraries were clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 high-throughput sequencing system instrument according to manufacturer’s instructions. The samples were sequenced using a 2 × 150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina’s bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.To assess RNA expression of genes in corneal tissue associated with SCCED, total RNA was extracted from corneal tissue gently debrided from four SCCED Boxer dogs with the eviously . Using t2 fold change| > 1.5 was considered significant [The datasets and transcript counts were estimated using Salmon (version 0.11.2) in quasi-mapping mode against dog reference genome CanFam3.1 with default settings . Salmon nificant .Ingenuity Pathway Analysis (IPA)  was used to evaluate key biologic pathways involving NOG to identify genes that could be impacted by altered NOG for further assessment with RNA Seq and immunohistochemical analysis.NOG deletion and three affected non-Boxer dogs without the NOG deletion. Corneal keratectomy specimens were preserved for future analysis and were fixed in 4% buffered paraformaldehyde overnight at 4 °C and then transferred to 70% ethanol before being embedded in paraffin. Tissues were sectioned at 5 μm and stained with hematoxylin and eosin. Immunofluorescence was performed following a previously described method with immunohistochemistry antibodies selected for the gene of interest and genes in the biologic pathway predicted to be impacted by altered gene function [Keratectomies were performed to treat the SCCED in five Boxer dogs with the function . In shorSeventy Boxer dogs either with, or having a documented history of SCCED and twenty-five Boxer dogs at least 10 years of age with no known history of SCCED were identified.KIF5C, NOTCH1, EMCS, Serpine2 and NOG.  but were pursued anyway because of the apparent involvement of the gene in the cornea. All remaining variants were in genes without a clear likely role in corneal disease and/or corneal wound healing and were SNPs or small indels located in a 3′ or 5′ untranslated, upstream, downstream or intronic region. These were not evaluated further.After filtering, 5013 variants were identified in at least 7 of 8 affected Boxers and in less than 1% of the 84 non-Boxer dogs from 17 different dog breeds. After filtering for genes identified as those likely to have a possible role in corneal disease and/or corneal wound healing, eight variants remained in five genes, KIF5C, NOTCH1 and EMCS were inconsistently found in the affected dog population and did not have a statistically significant association with SCCED. In contrast, the variants in Serpine2 and NOG were found in all of the SCCED Boxer dogs but also were found in 23 of 25, and 24 of 25 of the Boxers without SCCED, respectively.These eight variants were further evaluated by Sanger Sequencing. The variants in SERPINE2 variant was also found in 22 dogs of 11 breeds  while the NOG variant was found in only 4 dogs of one additional breed (Yorkshire Terrier) .When evaluated against the larger canine database of 391 non-Boxer dogs of 53 different breeds, the NOG variant was a 30 base pair deletion at a splice site . In silico splice site analysis predicted the NOG variant to disrupt a splice site as well as an enhancer motif site.The 2 fold change| > 1.5 considered significant, Serpine2, was not differentially expressed (0.015 log2 fold) between the SCCED groups while.With a cutoff of |logNOG expression was reduced (2.8 log2 fold).  compared to mean IHC scores from corneal samples from non-Boxer SCCED dogs without the NOG deletion  scores than corneal samples from non-Boxer SCCED dogs (Wilcoxon Test) although it was not found to be significantly different by RNA Seq. , which encodes the protein noggin, a constitutively-secreted, 46 kDa, disulfide-linked homodimer consisting of two, 206 amino acid polypeptide chains [NOG defect highly associated with SCCED Boxer dogs, but these NOG deficient dogs also had greater than two fold reduction of corneal epithelial noggin RNA signal and significantly reduced noggin staining in the stroma of keratectomy samples compared to NOG+ SCCED dogs. Noggin is involved in the bone morphogenetic protein (BMP) pathway where it serves as a potent inhibitor of BMP of the transforming growth factor-β superfamily. Noggin binds to BMPs receptor complex (BMPR-I) preventing its activation [Our data have demonstrated that many Boxer dogs with superficial nonhealing corneal ulcers have a genetic defect in the NOG defect, decreased tissue noggin, alterations in the BMP signaling pathway and delayed corneal epithelial wound healing in dogs diagnosed with SCCED. Several studies have also observed reduced keratinocyte proliferation and delayed dermal wound healing associated with reduced noggin and increased BMP activity [This study demonstrated a significant association between a genetic activity , 32. BMPactivity , 34. Limactivity , consistAlthough BMPs and Noggin effect on corneal wound healing has not been well described, BMPs and noggin have been shown to have an effect on dermal wound repair and healing. BMP2 and BMP4 have been demonstrated to reduce keratinocyte proliferation , 37 and NOG mutations have been reported in humans as an autosomal dominant disease with variable phenotype, commonly referred to as symphalangism spectrum disorder (NOG-SSD), involving 5 main autosomal dominant syndromes: (1) proximal symphalangism; (2) multiple synostoses syndrome 1; (3) stapes ankylosis with broad thumbs and toes; (4) tarsal-carpal coalition syndrome; and (5) brachydactyly type B2 [NOG deletion included in this study. type B2 –40. OtheNOG deletion. If noggin deficiency is associated with suppression of LEPC growth one might hypothesize that many Boxers will have a normal phenotype until they suffer a corneal abrasion and at that point the NOG deletion and its impact on the BMP signaling pathway could prevent the LEPC growth and normal healing that would occur in a dog without this deletion. Only at that time would the SCCED phenotype become apparent. Additionally, since the deletion is not one that would shorten life span or impact successful reproduction it would not have been naturally selected against and could be quite widespread within the breed. However, the pathogenesis of SCCED in dogs has been reported to a reduced adhesion of the corneal epithelium to the underlying corneal extracellular matrix and not a result of reduced corneal epithelial growth [In the dogs evaluated here, Boxer dogs without a known SCCED history also commonly had the l growth , 2, 17. l growth , leadingNOG) which was significantly associated with Boxer dogs with SCCEDs compared to non-Boxer dogs. Furthermore, RNA sequencing of corneal samples and immunostaining of keratectomy specimens from SCCED Boxer dogs deficient in NOG revealed reduced RNA signal and expression of NOG, respectively, and altered expression of BMP signaling pathway factors compared to WT dogs. A limitation of this study is that we compared corneal tissue from Boxer dogs with SCCEDs to non-Boxer dog with SCCEDs, rather than comparing it to corneal tissue from unaffected control dogs. Since SCCEDs are likely to have RNA and protein expression alterations that occur as a consequence of the corneal ulcer, comparison to unaffected corneal tissue could have emphasized changes that occur due to SCCED rather than the underlying cause. Finally, additional analysis of the NOG RNA for alignment and transcript analysis would have been useful but unfortunately the quality and quantity of NOG RNA from debrided cells was quite limited and prevented additional transcript analysis.In this study, using whole genome sequencing (WGS), we identified a 30 base pair deletion at a splice site in the noggin gene . Dysregulation of LEPC may play a role in the pathogenesis of RCE. These results suggest that NOG deficiency play a role in SCCEDs and RCEs. Further study of this defect in the pathogenesis of SCCED and RCE appears warranted.Many Boxer dogs with SCCED have a genetic defect in Additional file 1.


RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.Mutations in the gene for Retinitis Pigmentosa GTPase Regulator ( RPGR) [Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal diseases caused by a progressive degeneration of rod and cone photoreceptors that results in the eventual loss of vision. Mutations in more than 200 genes cause RP and collectively these affect one in 3000–5000 people, representing a major cause of inherited forms of blindness globally. Mutations causing X-linked RP (XLRP) are particularly severe with retinal disease presenting within the first few decades of life. The majority of mutations causing XLRP have been identified as loss-of-function alleles in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) –4.The RPGR protein is located within the photoreceptor connecting cilium , a membrRPGR generates multiple transcripts, and two major isoforms are well characterized [RPGRex1–19, is broadly expressed in multiple tissues, whereas a tissue-restricted RPGRORF15 is expressed primarily in retinal photoreceptors. The RPGRORF15 variant contains a unique 3ʹ terminal ORF15 exon that encodes two critical functional domains: a highly repetitive glycine/glutamic acid-rich domain and a C-terminal basic domain with homology to tubulins. The length of the repetitive region varies greatly among species and even among healthy human individuals. Furthermore, in-frame deletions that shorten this region by up to one third are well tolerated and appear to cause no functional deficits in mice [RPGR making it an attractive target for gene therapy [Alternative splicing of cterized , 12. The in mice , 14. Nev therapy .RPGR gene constructs are fraught with elevated risks due to the unique characteristics of the sequence. The repetitive region of RPGR is inherently unstable, difficult to sequence, and prone to spontaneous mutations during in vitro handling and subcloning [RPGR genes packaged into AAV vectors have later been found to contain many spontaneous changes in this repetitive region [RPGRORF15 can be targeted via CRISPR/Cas9 in induced pluripotent stem cells [While gene replacement therapies for RPGR-associated diseases have shown promise as a therapeutic strategy –21, engibcloning . Wildtype region  but the e region , 24, ande region –27. Receem cells , 29. CorRPGR OFR15 exon, we have proposed a simplified, variant version of gene editing. In this design, a double-stranded break in genomic DNA with CRISPR is first introduced at a target site in the repetitive region. Then the ensuing NHEJ would produce random base insertions or deletions that could in theory shift the reading frame in up to one third of cells thereby restoring expression of the critical C-terminal domain. While the therapy is not expected to restore gene structure in all target cells, based on previous clinical studies of retinal degenerative conditions and the known reserve capacity in retinal neurons, even a small fraction of surviving photoreceptors will support meaningful visual function and help maintain quality of life for the affected individuals. This design obviates the need for the rate-limiting and template-dependent homologous recombination step making it potentially feasible for in vivo intervention. As a proof-of-principle to treat XLRP caused by RPGR frameshift mutations, we chose the naturally occurring rd9 mouse model, which carries a 32-bp insertion in the ORF15 exon resulting in a frame shift that truncates RPGRORF15 protein [rd9 mice using CRISPR, delivered via subretinal injection of AAV vectors or pronuclear injection of sgRNA/SpCas9 ribonucleoprotein (RNP) complex, to restore RPGR function in somatic retinal and germline cells. We examined whether this approach could restore the full-length RPGRORF15 expression and alleviate the disease phenotype.Taking advantage of the unique features of the  protein , 30. We rd9 and C57BL/6 mice were used and for all experiments, rd9 littermates or age matched C57BL/6 litters were used for comparisons. Details regarding the age, number of animals analyzed, and blinding conditions are described in the respective figure and/or legend. Unless otherwise noted, animals were euthanized and tissue collected at 3–6 weeks of age.All procedures performed with animals were approved by the Animal Care and Use Committees at the National Institutes of Health and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Both male and female http://crispr.mit.edu/) and Benchling (https://benchling.com/). Several other sgRNAs were tested in the germline editing experiments but proved unsatisfactory. The sgRNA was synthesized with T7 in vitro transcription as described [rd9 mice (rd9/rd9) as described [The sgRNA target sequence (5ʹ-GAAGAGGGGGAGAGGAAGAA-3ʹ) was selected using the CRISPR design tool  or fixed in 4% paraformaldehyde/PBS. For samples in fixative, the anterior segment was removed after 5–10 min of fixation and the posterior eye cup was returned to fixative for several hours. Eye cups were then washed several times in PBS, embedded in 7% Type XI agarose, and cut into 75–100 μM cross sections using a vibratome. Frozen eyes were sectioned using a cryostat, collected on slides, and subsequently fixed in 1–4% paraformaldehyde immediately prior to antibody staining. All sections were blocked in 5% donkey serum (Equitech-Bio) in PBS-T for >1 h prior to incubation with antibodies diluted in blocking buffer. After overnight incubation in primary antibody, sections were washed several times in PBS before incubation with secondary antibody and DAPI for 1–4 h. Following several washes with PBS, sections were mounted in FluormountG (Southern Biotech). Slides were imaged on either a Zeiss LSM 700 or 880 confocal microscope and processed using Zen software. Maximum intensity projections were generated from z-stack images representing 2–8 μM of section thickness and for each dataset, the number of slices and imaging plane thickness were kept constant between treatment groups.n = 3 per genotype) were homogenized independently in RIPA buffer  containing 1X protease inhibitors (Roche). Lysate concentration was determined using a Pierce BCA protein assay kit (Thermo) and samples were equally loaded and separated on a 4–15% TGX Stain-Free SDS-PAGE gel (Bio-Rad). Following electrophoresis, separated proteins were wet transferred to PVDF for immunoblotting. Blots were incubated in StartingBlock PBS blocking buffer (Thermo) for >1 hr prior to incubation with antibodies diluted in blocking buffer. After overnight incubation in primary antibody, blots were washed several times in PBS before incubation with secondary antibody for approximately 1 h. After several PBS washes, blots were incubated with substrate  and digitally imaged on a Bio-Rad ChemiDoc imaging system.Following euthanasia eyes were removed, and the retina was dissected from the eye and snap frozen on dry ice. Single retinas (Antibodies used that were custom generated in our laboratory included RPGR-s1 (rabbit), RPGR-570 (rabbit), and c100 (rabbit), S-Opsin (chicken), and Rootletin (chicken). Other antibodies included Rhodopsin 1D4 , M-Opsin , GT335 , AHI1 , and Glial Fibrillary Acidic Protein .Dual AAV vectors were generated: one containing a Streptococcus pyogenes Cas9 (SpCas9) driven by the rhodopsin kinase (RK) promoter  and anotn > 18) were anesthetized via IP injection of ketamine/xylazine and eyes were dilated and topically anesthetized via drops of 0.5–1% tropicamide, 2.5% phenylephrine hydrochloride, and 0.5% proparacaine. A small incision was made through the cornea anterior to the limbus and a blunt needle fitted to a Hamilton syringe was inserted to deliver ~1 μl of each AAV (7.5e9–1e10 vg/μl) diluted in PBS/fluorescein, to the subretinal space. Animals were euthanized 10–12 weeks post injection and the eyes collected for analysis. A larger sample size was chosen for analysis based on the variability observed in preliminary dose–response experiments. Because each mouse received both RPGR-targeted (right eye) and control non-targeted AAV (left-eye), treatment group randomization was not necessary.Mice  using the following thermocycling conditions: one cycle of 98 degrees for 2 min; 35–40 cycles of 98 degrees for 10 sec, 68 degrees for 45 sec; and a final cycle of 68 degrees for 1 min. PCR product was sequenced by Genewiz using either Sanger or PacBio deep sequencing platforms. For PacBio sequencing, the PCR amplicon was generated from a pool of three retinas from either RPGR-targeted or non-targeted (GFP) AAV-injection. CCS analysis of the single pass non-consensus raw data was performed by Genewiz. Returned reads were further filtered in Matlab 9.4 for alignment to a reference rd9 ORF15 sequence.Genomic DNA isolated from either tail biopsy or retina was used as a template to amplify a region of rd9 mutant allele in mice by injecting the constructs into fertilized mouse embryos. A 20-bp target sequence was selected for the sgRNA that included part of the native ORF15 sequence and the first 8 bp of the mutant duplication in rd9 , RPGRORF15 protein was observed in patches of photoreceptors throughout the retina  further demonstrated germline mosaicism of the founders with ≈18% of female progeny receiving the edited allele  and hemizygous rd9*  mice was expressed at levels comparable to controls  promoter [rd9 ORF15 sgRNA that also contained a tdTomato reporter. An irrelevant target sgRNA (GFP) was used as a negative control. Subretinal injections were performed in rd9 mice and tissues were collected 8–12 weeks post injection for analysis. Confocal images of RPGR-570 antibody co-stained with a marker to the ciliary rootlet demonstrated expression of RPGRORF15 at the connecting cilium in a subset of ORF15-treated photoceptors  containing a single precise 32-bp repeat of the ORF15 repetitive region . Future efforts, therefore, should be directed at better understanding the molecular events following CRISPR-mediated DNA breaks in the RPGR gene and at improving overall AAV transduction efficiency to achieve a higher rate of gene correction. Recent reports [RPGR. Deep sequencing was performed on AAV-treated retinas to capture the range of gene modifications. Perhaps unsurprisingly, the ORF15 amplicon proved difficult to sequence, despite multiple attempts and platforms, and heterogeneity in the sequences obtained from both ORF15 targeted and control samples precluded confirmation of precise edits. Nevertheless, isoform-specific antibodies were used to show unambiguously that full-length RPGR-ORF15 protein was restored in rd9 retina following treatment and that the protein was glutamylated and localized correctly to the connecting cilia. Finally, one of the main limitations of the CRISPR/Cas9 system is that deleterious off-target mutations can occur which could pose potential risks in clinical applications. Next-generation sequencing could detect such off-target mutations although sensitivity may be low when such mutations are rare. Prediction-based methods offer higher sensitivity of detection using target-specific DNA amplifications. These assays would help determine the safety profiles of the chosen CRISPR design. Because RPGR ORF15 sequences are poorly conserved between mice and humans, future choices of CRISPR guide RNA sequences will substantially differ as future studies transition to human cell-based models. In the current study in mice, we did not assess off-target effect of the CRISPR/Cas9 and cannot make any statement about off-targets of the CRISPR design in mice. It should be emphasized, however, that careful evaluation of off-target mutations, perhaps in human iPSC-derived retinal organoids, is critical to establish the safety profiles of this strategy.Our study is the first to demonstrate template-free in vivo correction in mature photoreceptors as a proof-of-principle for future work that aims to correct disease-causing mutations in  reports  that NHEORF15 expression in patches of photoreceptors throughout their retinas which served as proof that these were genuinely chimeric founders. Moreover, the rate of transmission of the genome-edited allele was about 10–15%, which indicated a degree of genetic mosaicism as would be expected in the founders and was in sharp contrast with expected outcomes of contamination by wild type or heterozygous parental breeders. In view of the highly repetitive sequence context, our findings raised the intriguing possibility that deletion of the 32-bp segment was mediated by an alternative DNA repair mechanism such as microhomology-mediated end joining (MMEJ) [rd9 allele back to a correct reading frame and effectively rescues the disease-causing mutation. This hypothesis provides a plausible explanation for our unexpected finding in germline manipulated mice, and it would be very important to further validate this hypothesis in future experiments in mouse as well as human cells and tissues. As compared to NHEJ, an MMEJ driven process offers the exciting prospect that repair could be achieved at levels above the one third threshold, since the end joining would not be a random event. It would be important for future studies to take advantage of human iPSC-derived retinal organoids to test this approach on human mutations in RPGRORF15. Such studies should ultimately determine if a simplified genome editing strategy as outlined in this work can become a legitimate future therapy for X-linked RP that is both safe and effective.An intriguing observation emerged from this study that suggests NHEJ may not be the only mechanism that mediates end joining and repair in the ORF15 exon. We were initially surprised to find that both founders born from independent batches of microinjections carried the same modification, which turned out to be a 32-bp deletion near the site of DNA breaks. We have conducted rigorous control experiments and evaluated breeding schemes ruling out any possibility that this could be due to cross contamination or mixing of mouse strains. Most tellingly, the founders showed restored RPGRg (MMEJ) , 40. Theg (MMEJ) , 41. In


Radiomics is a promising technique for discovering image based biomarkers of therapy response in cancer. Reproducibility of radiomics features is a known issue that is addressed by the image biomarker standardisation initiative (IBSI), but it remains challenging to interpret previously published radiomics signatures. This study investigates the reproducibility of radiomics features calculated with two widely used radiomics software packages  in comparison to an IBSI compliant software package (PyRadiomics). Intensity histogram, shape and textural features were extracted from 334 diffusion weighted magnetic resonance images of 59 head and neck cancer (HNC) patients from the PREDICT-HN observational radiotherapy study. Based on name and linear correlation, PyRadiomics shares 83 features with IBEX and 49 features with MaZda, a sub-set of well correlated features are considered reproducible . We explore the impact of including non-reproducible radiomics features in a HNC radiotherapy response model. It is possible to classify equivalent patient groups using radiomic features from either software, but only when restricting the model to reliable features using a correlation threshold method. This is relevant for clinical biomarker validation trials as it provides a framework to assess the reproducibility of reported radiomic signatures from existing trials. Radiomics4 is the high-throughput extraction of image features from standard-of-care medical images, with the hypothesis that macroscopic image features offer insight into disease process at a molecular level5. Radiomics analysis has been widely adopted in oncology research, showing potential to identify magnetic resonance image (MRI) based biomarkers for clinical outcomes in head and neck cancers (HNC)6. The evolution of radiomics features during treatment, commonly referred to as delta-radiomics, may offer more information than a single time point to identify biomarkers during radiotherapy or chemotherapy and has been explored in HNC with CT imaging10, positron emission tomography (PET) imaging11 and more recently in MRI studies16.Extracting textural features from medical images provides additional information6 details the investigation of MRI radiomics features for applications such as image segmentation, histopathological classification and prognostic or predictive biomarkers. Previous HNC studies explore a range of MRI sequences, from anatomical imaging such as T1 weighted, T2 weighted and short tau inversion recovery (STIR) to functional imaging such as diffusion weighted (DW-MRI) and dynamic contrast enhanced imaging (DCE-MRI). This study focuses on radiomics features calculated on apparent diffusion coefficient (ADC) maps derived from DW-MRI images. The apparent diffusion coefficient has been correlated to cellularity in many tumour types17 and has been linked to cell proliferation in head and neck squamous cell carcinoma18. During radiotherapy, early changes in ADC have been linked to treatment response outcomes for multiple tumour types, making it a potential candidate for biological image-guided adaptive radiotherapy on MRI guided radiotherapy systems19.A review of head and neck cancer studies5, with the variability of each task known to impact model performance22. In MRI studies, these effects have been investigated with regard to image acquisition27, region of interest segmentation30, image pre-processing32, feature extraction35 and feature reduction combined with classifier training37. To address the known variability issues of features extracted with different software35 the image biomarker standardisation initiative (IBSI)38 has proposed a set of feature extraction guidelines. In head and neck cancer, MRI studies have reported feature extraction with software such as MazDa41, IBEX42 and in-house solutions based on MATLAB49, none of which adhear to the IBSI guidelines.Developing a radiomics model is often considered as a series of discrete tasks each with its own challenges51 select a small set of features based on previously reported radiomic signatures. The IBSI guidelines should mitigate known feature reproducibility issues35 in future studies, but feature uncertainty remains a problem when interpreting previously reported radiomic signatures. This study investigates the correlation between features generated with open-source radiomic software packages (IBEX52 and MaZda53) used in many published studies against an open-source tool (PyRadiomics54) which follows the IBSI guidelines. We then explore the impact of non-reproducible radiomics features on a HNC radiotherapy response model using through therapy ADC radiomics features. Our comparison focuses on DW-MRI of head and neck cancer but provides general confidence on which previously reported radiomics features can be reproduced with software that adheres to the IBSI guidelines.Radiomic analysis generates hundreds of features, making feature reduction a crucial step to prevent overfitting when developing a radiomics model. Validation studies59, 16 GLRLM and 5 NGTDM features. The IBEX feature set had  7/13 intensity histogram, 6/18 shape, 18/21 GLCM (at three neighbourhoods), 11/11 GLRLM and 5/5 NGTDM features. The MaZda feature set had  6/14 intensity histogram, 6/22 shape, 10/12 GLCM (at three neighbourhoods) and 7/7 GLRLM features. Shared features that had dissimilar names included shape features , , , , , , GLCM features , , , ,  and one GLRLM feature .To investigate the impact of feature variability on patient modelling, 36 patients with imaging at all eight time points were clustered into two patient groups using an unsupervised learning method. Radiomic features were calculated throughout radiotherapy on apparent diffusion maps coefficient (ADC) maps, derived from diffusion weighted MRI. Hierarchal clustering based on all shared radiomics features Fig. a resulte35 explore a range of image modalities and feature extraction software. A study of mammograms and HNC computed tomography images33 also extracted IBEX and MaZda features, but compared them against one another and with two in-house software packages. One feature extraction study of HNC patients was less comparable as it analysed PET images35 and compared two in-house radiomics software packages. A study of HNC patients who had both CT and MRI imaging34 also extracted PyRadiomics features but compared them to features extracted with Moddicom55 and the radiomics extension to CERR56. Whilst that study34 also extracted features from MRI images of HNC patients, the features were from T2 weighted images, whereas our study is the first, to our knowledge, to investigate feature extraction stability on ADC maps from diffusion weighted MRI.Existing studies of variability in radiomics feature extraction33 demonstrated high intra-class correlation (ICC) of intensity histogram features and low ICC of GLCM features, though this was partially attributed to the use of default GLCM extraction settings for each software. The study of HNC patients with both CT and MRI images34 showed that CT features had high Spearman correlation of intensity histogram, shape and GLCM features and lower ICC for GLRLM and grey-level size zone matrix (GLSZM) features. The T2 weighted MRI features compared in that study had high Spearman correlation for intensity histogram and shape features between PyRadiomics and CERR and only shape features highly correlated between PyRadiomics and Moddicom; the lack of correlation with intensity histogram features was attributed to Moddicom performing an image intensity correction on the T2 weighted MRI. In our study NGTDM features were extracted with IBEX and had a mixture of well correlated (1/5) and uncorrelated (4/5) features, the previously mentioned PET study35 also extracted NGTDM features but only a percentage of grouped textural features above an ICC threshold was reported. The similarity of our findings with those previously reported may indicate that variability in radiomics features due to the extraction software can be considered independent of the imaging modality.Our study observed a similar trend of feature extraction reproducibility to existing studies; intensity histogram features have high reproducibility across feature extraction software, with shape and textural features being more software package dependent. The study of mammograms and HNC CT images35 have investigated the impact of feature extraction variability on HNC model performance. The joint MRI and CT imaging study34 performed hierarchical clustering of patients for each feature class separately  and observed consistent clustering of clinical variables  for radiomics feature classes with a high reproducibility. The PET study35 demonstrated that models of local tumour control built on features from two different radiomics packages can stratify patients into very similar low or high risk recurrence groups, with the model features being highly correlated between the two software packages (ICC > 0.9). These studies and our results demonstrate that it is possible to generate equivalent radiomics models with different feature extraction software, but that care must be taken to ensure that features are highly correlated between the software packages. Additionally, our study extends this observation to patient classification based on radiomics features prior to treatment, at multiple time points during treatment and post-treatment.Two radiomics feature reproducibility studies35 is the ambiguity in matching features between different software, firstly identifying shared features and secondly matching the feature extraction settings which often differ between software package. Name similarity does not guarantee equation similarity, for example the shape feature “SurfaceVolumeRatio” is calculated with a voxel based volume in IBEX and a mesh based volume in PyRadiomics. Equation similarity can be difficult to establish due to different notations, such as the formulation of the shape feature “SurfaceArea” between PyRadiomics and IBEX  was selected for this study as it supports batch feature extraction, from the software documentation it was inferred that there was minimal change to the feature extraction code from earlier MaZda versions. The region of interest was calculated with a Plastimatch derived label map for PyRadiomics and MaZda and was calculated directly from the DICOM structure file (RTSTRUCT) by IBEX, which may have introduced variability in the image region used for feature extraction. We explored radiomics features calculated on the original ADC map to avoid additional variability from image filtration  between software implementations. Image filtration reproducibility between software using MRI images may be worth investigating as differences in wavelet filtered features from PET images have been previously reported58. This study reports the variability of radiomics features extracted from ADC maps, that are a relatively quantitative image, generating features not significantly influenced by scanner manufacturer or magnetic field strength26; radiomics features extracted from T1 and T2 weighted images are less reproducible across scanner and not recommended at this point for multicentre trials24. The increasing uptake of MRI for simulation and therapy in radiotherapy departments offers an unprecedented opportunity to characterise tumour response and personalise patient treatment. This study provides information on how feature extraction software can impact the reproducibility of a radiomics workflow, which we should endeavour to optimise in order to accelerate discovery through reproducible research and data sharing.Magnetic resonance imaging offers insight into disease related anatomical and functional changes and is ideal for radiomics analysis as multiple MRI sequences can provide complimentary informationThis work highlights feature and model reproducibility issues due to different radiomic analysis software. We propose a correlation threshold method to select reproducible features and demonstrate that the identified features from both software generate an equivalent model. This is relevant for the selection of radiomic features in clinical biomarker validation trials as it provides a framework to assess the reproducibility of radiomic signatures from existing studies.59 , date of enrolment of the first participant 30/05/2017. All study participants were 18 years and older, informed consent was obtained from all participants.The PREDICT-HN study61 sequence . Apparent diffusion coefficient maps were calculated from the DWI images using the default mono-exponential model.MRI imaging was performed on a Siemens 1.5 T Aera scanner to acquire both anatomical and functional images. The gross tumour volume (GTV) was contoured on pre-treatment T2 weighted turbo-spin-echo (T2w-TSE) images  by a radiation oncologist. Throughout treatment, the pre-treatment contours were rigidly registered to the through treatment images and manually adjusted to anatomical boundaries, then propagated onto the apparent diffusion coefficient (ADC) maps and visually verified. Diffusion weighted images (DWI) were acquired with the BLADE62), grey-level run length matrix (GLRLM63) and neighbourhood grey-tone difference matrix (NGTDM64) features were calculated on the original ADC map only; the reproducibility of image filtration prior to feature extraction was considered outside the scope of this study. The region of interest for feature extraction was calculated directly from the DICOM radiotherapy contours (RTSTRUCT) with IBEX and an intermediate step to convert the DICOM contours to a binary label map with Plastimatch (version 1.7.3)65 was required for PyRadiomics and MaZda. The feature extraction settings  using PyRadiomics (version 2.1.0), IBEX (version 1.0 Beta) and MaZda (qmazda 19.02). All available intensity histogram (IHIST), shape, grey-level co-occurrence matrix (GLCMTo determine the relationship between features generated with the established radiomics software (IBEX/MaZda) and the IBSI compliant software (PyRadiomics) we performed a linear regression analysis on radiomics features extracted from ADC maps of all patients at all time points. A linear fit was calculated between PyRadiomics and IBEX or MaZda for every feature in a given feature class . Features with an invalid value (i.e. infinity due to division by zero) were excluded during the linear regression. A list of shared radiomics features was collated by identifying features extracted by both PyRadiomics and the alternative software package, and was based primarily on name and equation similarity. A small number of features that showed a high linear correlation but had dissimilar names, for example ‘SurfaceArea’ and ‘Perimeter’, were also included. The list of shared features, feature correlations and the number of images used per feature correlation can be found in Supplementary Tables 67 using Ward's minimum variance clustering method68 on scaled radiomics features (z-score standardisation) with an automatic minimum clustering threshold to generate no more than two clusters.To demonstrate the potential impact of incorporating non-reproducible features in a radiomics model we used unsupervised learning to identify two groups of patients, based on radiomic features at pre-treatment, throughout radiotherapy and post-radiotherapy. Separate radiomics models were generated based on PyRadiomics and IBEX/MaZda features, first using all features and then with the sub-set of reproducible features. Reproducible features were selected as those with a high Pearson’s correlation coefficient (To test the sensitivity of clustering to the selected correlation threshold, we performed the clustering over a range of thresholds (Supplementary Information.


Distinct but related species of elephant endotheliotropic herpesviruses (EEHVs) circulate within Asian and African elephant populations. Primary infection with EEHVs endemic among Asian elephants can cause clinical illness and lethal EEHV hemorrhagic disease (EEHV-HD). The degree to which this occurs among African elephants has not been fully established. Recent cases of EEHV-HD caused by the EEHV3 species in African elephants housed in North American zoos has heightened concern about the susceptibility of this elephant species to EEHV-HD. In this study, we utilize the luciferase immunoprecipitation system (LIPS) to generate a serological assay specific for EEHV3 in African elephants by detecting antibodies against the EEHV3 E34 protein. The results showed that the majority of tested elephants from four separate and genetically unrelated herds, including five elephants that survived clinical illness associated with EEHV3, were positive for prior infection with EEHV3. However, African elephants who succumbed to EEHV3-HD were seronegative for EEHV3 prior to lethal infection. This supports the hypothesis that fatal EEHV-HD caused by EEHV3 is associated with primary infection rather than reactivation of latent virus. Lastly, we observed that African elephants, like Asian elephants, acquire abundant anti-EEHV antibodies prenatally and that anti-EEHV3 specific antibodies were either never detected or declined to undetectable levels in those animals that died from lethal disease following EEHV3 infection.IMPORTANCE Prior to 2019, only five cases of clinical disease from EEHV infection among African elephants had been documented. Since 2019, there have been at least seven EEHV-HD cases in North American zoos, resulting in three fatalities, all associated with EEHV3. Evidence is accumulating to suggest that EEHV-associated clinical illness and death among Asian elephants is due to primary infection and may be associated with waning anti-EEHV antibody levels in young elephants. The development of the EEHV3 serological test described in this study enabled us to confirm that similar dynamics may be contributing to EEHV-HD in African elephants. The ability to screen for EEHV immune status in African elephant calves will have a major impact on managing captive African elephant herds and will provide new tools for investigating and understanding EEHV in wild populations. Elephas maximus) and African elephants (Loxodonta africana) (Elephant endotheliotropic herpesvirus (EEHV) can cause lethal EEHV hemorrhagic disease (EEHV-HD) in both captive and wild elephants , 2. Althfricana) , 4, the fricana) . Recent fricana) . Due to fricana) , suggestProboscivirus genus-specific proteins. The majority of EEHV-HD cases in captive African elephants have been caused by a single GC-rich species, EEHV3  a family herd of 11 elephants in which at least 5 herdmates have demonstrated EEHV3 shedding or viremia, suggesting that most or all herd members have been exposed and are chronically infected , and ii Table 2Table 2. Gaussia luciferase (GLuc) protein for the LIPS assay, as described previously (P = 0.2) (data not shown). To further validate the assay, we screened the serum samples from the EEHV-positive and EEHV-HD group I cohorts against the E. maximus interleukin 4 (IL-4) -GLuc fusion protein in the LIPS assay, which yielded results similar to those for the no-serum controls  adult elephants from three different herds with unknown EEHV history for some of the individuals  and (ii)18–We are unaware of any previous studies investigating the maternal-to-fetal anti-EEHV antibody transfer in African elephants. However, studies in Asian elephants suggest that the majority of antibody transfer occurs prenatally and includes anti-EEHV antibodies , 21. HavThe results presented here provide compelling evidence that primary infection with EEHV3 can underlie fatal EEHV-HD in African elephants . We alsoThe EEHV-specific LIPS assay has several key features that support its use in investigating EEHV in African elephants. These features include the following: (i) only small amounts of serum are needed for testing, (ii) species-specific reagents are not required, and (iii) it provides the ability to rapidly produce and screen new antigens. Thus, the LIPS assay system provides a flexible platform to monitor EEHV infection status and to explore EEHV biology in African elephants. The adaptability of LIPS was demonstrated when we identified the E34 antigen, which is both highly immunogenic in elephants and divergent between EEHV species. By incorporating E34 into the LIPS assay, we were able to distinguish between infection with EEHV3 and the other EEHV strains  in African elephants. The ability to differentiate between strains is critical to address the knowledge gaps regarding EEHV in African elephants, as well as to determine whether previous cases of EEHV infection were due to primary infection or reactivation.n = 28). Although the assays showed 100% sensitivity and specificity, we anticipate that surveillance of larger numbers of animals will reveal a range of antibody responses in elephants with evidence of chronic infection. Whether this will alter the cutoff value for our EEHV3 E34 serological assay remains to be determined. Our study is also the first to document transplacentally acquired anti-EEHV antibodies in African elephants. Although our study provides evidence for the acquisition and disappearance of maternally transferred EEHV antibodies in some individuals across elephant species, additional juvenile animals will need to be surveyed to determine whether this is broadly representative of most elephants. It seems fairly clear that being seronegative toward a specific EEHV type renders young elephants from both elephant species susceptible to severe primary EEHV infections, leading to death.In this study to characterize the specific seroprevalence of EEHV in African elephants, we found that 100% of African elephants tested had evidence of prior EEHV infection. One limitation of this research is the small number of adult and juvenile animals tested , two of those elephants began to show seroconversion for E34 during their clinical illnesses with EEHV3-HD . HoweverUse of the LIPS assay to identify potentially EEHV3-seronegative African elephants, which we now show may be associated with the development of lethal EEHV-HD cases in African elephants, could be an important tool in elephant management. Serostatus can be taken into account when assessing the risk of transferring elephants between facilities. Additionally, in cases in which low-level viremia is detected, serostatus and other clinical data can be used to determine whether to initiate treatment for potential EEHV disease. These treatments often involve a number of disruptive, costly, and stressful activities for both zoo personnel and the elephant herd. Lastly, this assay will be a necessary tool to evaluate the efficacy of future candidate vaccines against EEHV.Serum samples were obtained from 11 African elephants at the San Diego Zoo Safari Park (SDZSP). At least 5 of the elephants had evidence of latent EEHV infection from either an EEHV-positive trunk wash or viremia  . Sera weMN373268). Similarly, the genomic sequences for EEHV3B, EEHV2, and EEHV6 were determined . The U39 sequences for EEHV2 and EEHV6 were described previously  for storage up to 1 month at 4°C. For use in the assay, samples were diluted 1:5 in the same buffer. Luminescence units (LU) of stored GLuc extracts were determined on the day of the assay, and a master mix of each GLuc extract containing 1 × 107 LU per 50 μl was made. Fifty microliters of GLuc extract master mix was added to 50 μl of diluted serum (in duplicate) and incubated for 1 h at room temperature on a shaker. After 1 h, GLuc extract-diluted serum was added to 5 μl of a 30% protein A/G-bead suspension  in a 96-well filter plate (Millipore). Samples were incubated with the beads for 1 h at room temperature on a shaker. Beads were then washed five times with buffer A, followed by two PBS washes, using a 96-well vacuum manifold (Millipore). An OmniStar luminometer (BMG Labtech) was used to inject 50 μl of Renilla luciferase assay substrate into each well, shake the plate for 2 s, and then record relative light unit (RLU) values for 5 s. Values reported represent the average RLUs over 5 s postinjection.LIPS analysis was performed as described previously , 24. BriGaussia fusion proteins, supernatants or extracts from cell-associated GLuc fusions were mixed with 2× sample loading buffer and resolved by SDS-PAGE, and immunoblotting was carried out as described previously  was used to detect the EEHV-GLuc fusion proteins.For analyses of EEHV eviously . A commeU tests, and significance was set at P values of ≤0.05. The cutoff value for determining sensitivity and specificity for each viral antigen was derived from the mean antibody titer of the uninfected samples plus 5 SDs.Statistical analysis was performed with GraphPad Prism software, version 8.4.3. In general, LIPS assays were performed in duplicate, with at least two independent experiments for each sample. Results for different treatment groups are presented as geometric means ± standard deviations (SDs). Group means were compared with Mann-Whitney MZ269261, MZ269260, and MZ269262, respectively.The E34 sequences derived for EEHV3B, EEHV2, and EEHV6 were deposited in GenBank with accession numbers


Seizures are a prominent feature in N-Methyl-D-Aspartate receptor antibody (NMDAR antibody) encephalitis, a distinct neuro-immunological disorder in which specific human autoantibodies bind and crosslink the surface of NMDAR proteins thereby causing internalization and a state of NMDAR hypofunction. To further understand ictogenesis in this disorder, and to test a potential treatment compound, we developed an NMDAR antibody mediated rat seizure model that displays spontaneous epileptiform activity in vivo and in vitro. Using a combination of electrophysiological and dynamic causal modelling techniques we show that, contrary to expectation, reduction of synaptic excitatory, but not inhibitory, neurotransmission underlies the ictal events through alterations in the dynamical behaviour of microcircuits in brain tissue. Moreover, in vitro application of a neurosteroid, pregnenolone sulphate, that upregulates NMDARs, reduced established ictal activity. This proof-of-concept study highlights the complexity of circuit disturbances that may lead to seizures and the potential use of receptor-specific treatments in antibody-mediated seizures and epilepsy. Sukhvir Wright et al. present a NMDAR antibody-induced model of encephalitis in rats and use in vitro, in vivo, and in silico electrophysiology to examine alterations in neural circuit behavior. Their results suggest that reduction of NMDARs leads to increased excitability and seizure activity, and highlights the potential use of receptor-specific treatments in antibody-mediated seizures and epilepsy. As a result, the mainstay of treatment is immunotherapy that aims to reduce the levels of circulating neuronal autoantibodies or halt their production5. These treatments can be slow to work, and carry the risk of harmful side-effects as the healthy immune system is also compromised6. It is clear that the reduction of NMDARs in NMDAR antibody encephalitis causes a myriad of severe neurological symptoms, and that reversal of this pathological effect in the acute and even chronic stages of the disease has the potential to improve clinical outcomes9. However, as yet, there are no immune-sparing treatments that ameliorate the synaptic and circuit effects  caused by the NMDAR hypofunction11. The therapeutic potential of a receptor-specific treatment could be particularly beneficial in children who are affected during crucial developmental stages, marked by neuronal network plasticity including that of the NMDAR subunits13. Indeed, studies have found that after recovery children have persistent cognitive problems and fatigue, resulting in lower academic achievement and poorer quality of life15.Both in vivo and in vitro studies have clearly established that the pathogenic action of antibodies in N-methyl-D-aspartate receptor (NMDAR) antibody encephalitis is to bind to the surface of the NMDARs, cross-link the proteins and cause NMDAR internalization, resulting in a state of NMDAR hypofunction16. Seizures present as part of the disease course in >80% of patients and EEG changes are seen in >95%, more commonly than Magnetic Resonance Imaging (MRI) brain changes, as noted repeatedly in cohort and case studies17. Despite the symptom predominance of seizures in humans, producing reliable and consistent spontaneous seizure animal models of this disorder using passive transfer of human-derived antibodies is challenging20. In our previous passive transfer mouse model, we demonstrated increased in vivo seizure susceptibility (and therefore network hyperexcitability) 48 h after intracerebroventricular NMDAR antibody injection compared to controls, however spontaneous seizures were not seen21.While neuropsychiatric features are the most common presenting symptom of NMDAR antibody encephalitis, acute symptomatic seizures and encephalopathic electroencephalogram (EEG) changes are essential clinical and investigative features in patients of all ages24. Given the specific involvement of the excitatory NMDAR, it is tempting to speculate that the pathophysiology of these epilepsies diverges from a simple ‘excitation-inhibition’ imbalance and that an NMDAR-specific treatment may be more effective in minimizing acute symptoms such as seizures, and could potentially be given alongside immunotherapy to achieve more rapid symptomatic relief.Importantly, seizures within the context of NMDAR antibody encephalitis do not respond well to standard anti-seizure medications, as seen in other forms of antibody-mediated encephalitisThis study aimed to understand the causative changes in synaptic physiology contributing to NMDAR antibody-mediated seizures, and to explore the unmet clinical need for receptor-specific treatment. We developed a juvenile Wistar rat model of NMDAR antibody encephalitis, in which NMDAR antibodies derived from human patients were used to induce a robust and repeatable seizure phenotype. Combining in vitro, in vivo, and in silico approaches we have demonstrated that, counterintuitively, the epileptic dynamics emerge from reductions in excitatory neurotransmission caused by NMDAR antibodies, and provide proof-of-concept in vitro evidence of a NMDAR specific neurosteroid rescue treatment both in the rodent model, and in human epileptic brain tissue.18. The florescence intensities of NMDAR antibody injected and infused brain slices were significantly higher than those measured after injection or infusion of control human IgG or non-brain reactive monoclonal antibodies   injection, juvenile rats injected with NMDAR antibodies exhibited sustained and repetitive hyperexcitable behaviour including myoclonic twitches, jerks and jumps . Following the observations above, a depth electrode was placed in the CA3 region. Spontaneous epileptiform events were observed in the NMDAR antibody infused animals in vivo and evident in the EEG tracings . The greatest increase in power, seen in the 12–30 Hz range , was similar to the typical excessive beta activity observed in EEGs of NMDAR antibody patients (14–20 Hz)26.To demonstrate ictogenesis in vivo, 7-day osmotic pumps were used to deliver NMDAR antibodies or control antibodies into the lateral cerebral ventricles of juvenile Wistar rats . In line with the spontaneous epileptiform activity seen in vivo, there was a considerable increase in the number of interictal events and a reduced IEI of ictal spikes in NMDAR antibody infused animals compared to controls Fig. . CA3 hip27) to EEG power spectral densities, first for control animals, then for NMDAR antibody animals under different empirical priors with model fits for the power spectral densities shown for the final, selected model in Fig. We tested whether the in vitro changes in single neuron behaviour associated with NMDAR antibody contributed to in vivo  EEG patterns by using a computational model of microcircuit dynamics. Specifically, we tested the hypothesis that the microscale recordings can inform models of LFP dynamics using Bayesian model comparison of models with, and without prior information derived at the microscale. We first generated quantitative predictions of the population-level effect of NMDAR antibody based on in vitro differences between NMDAR antibody and control antibody exposed neurons measured using whole-cell patch clamp recordings Fig. . We thencontrol antibody and NMDAR antibody  conditions captures the main effect of NMDAR antibody infusion, which we know from the preceding results in a higher seizure likelihood, and is thus epileptogenic in the animals . (ii) The synaptic parameter difference derived from the NMDAR antibody , and the NMDAR antibody  conditions captures the main effect of transition into the seizure state, and thus represents ictogenesis .We then considered the overall winning model highlighted in Fig. control and NMDAR antibody  condition the ‘epileptogenesis’ parameters since they capture the difference between brain dynamics in the non-epileptic control animals and the animals with NMDAR antibody induced spontaneous recurrent seizures. Similarly, we consider the parameter differences between NMDAR antibody  and NMDAR  the ‘ictogenesis’ parameters since they capture the difference between brain dynamics in the interictal and ictal state. We derived initial parameterisations by fitting microcircuit models to the three empirically observed brain states as described above , but did not affect the amplitude  in control slices.We hypothesised that increasing NMDAR expression could rescue the electrophysiological features associated with NMDAR antibody. The neurosteroid pregnenolone sulphate (PregS) has a membrane delimited action to increase the trafficking of functional NMDARs to the neuronal membraneNext we tested whether these effects would rescue the NMDAR antibody induced abnormalities observed in vitro. We recorded LFP discharges in vitro before and after addition of PregS to brain slices from animals treated with NMDAR antibody or control antibody for 48 h or 7 days Fig. . The numn = 12; p = 0.5, Wilcoxon ranked pairs test), mini-IPSC amplitude  or mini IPSC IEI .We confirmed that the reduction in LFP spiking activity was associated with a rescue of the synaptic abnormalities by performing whole-cell patch clamp recordings in hippocampal slices from animals exposed to short (48 h) or more chronic (7 days) NMDAR antibody exposure. In both conditions, PregS increased the frequency of sEPSCS recorded in CA3 neurons from NMDAR antibody treated animals back to control levels , or in the IEI .Finally, we showed that this acute reversal of NMDAR antibody mediated electrophysiological effects by PregS could in principle be translated to human brains. Paediatric brain slices prepared from tissue resected during epilepsy surgery Fig.  was expo29. Combining electrophysiological recordings in a rat model of NMDAR antibody encephalitis and computational modelling of brain microcircuit function, we show that an effective reduction in excitatory synaptic neurotransmission observed at the single-neuron scale underlies NMDAR antibody epileptogenesis by altering the dynamical makeup of brain microcircuitry, making it more susceptible to excursions into pathological brain states. We fitted synaptic parameters of microcircuit models to spectral features of LFP data in different experimental conditions corresponding to a non-epileptic, interictal condition (control), an epileptic and interictal condition  and an epileptic and ictal condition . This allowed the separation of two main effects—epileptogenesis, and ictogenesis. In terms of parameter space, these effects were separable, meaning that they acted on different subsets of synaptic parameters. The transition from interictal to ictal state (i.e. ictogenesis) was best explained by changes in synaptic coupling that effectively push excitation-inhibition out of balance into a hyperexcitable state. Yet the pathophysiological process of epileptogenesis—i.e. the synaptic changes that render the brain more likely to undergo an interictal-ictal transition—are separable from those associated with ictogenesis. In fact, in our particular animal model we can show that the hypoexcitation induced by NMDAR antibody counterintuitively contributes to epileptogenicity, by increasing the sensitivity of microcircuits to fluctuations of the ictogenicity parameters. We demonstrate that this phenotype can be rescued with the neurosteroid, PregS, which, among other pharmacological actions, is known to increase NMDAR availability at the neuronal membrane. Furthermore, we reproduced the synaptic effect of PregS in human brain tissue in vitro, indicating its potential use as precision-treatment for epileptic seizures in patients with established NMDAR antibody encephalitis and potentially other seizure aetiologies associated with NMDAR-hypofunction.NMDAR antibodies disrupt normal brain circuit function and are associated with a range of neurological symptoms in patients, including epileptic seizures. The majority of these symptoms are now understood to be directly caused by antibody-induced hypofunction of the excitatory NMDAR. However, the link between reduced NMDAR function and increased seizure propensity has remained unexplained, and is particularly counterintuitive from a classical perspective of seizures arising from an excitatory-inhibitory imbalance31. Juvenile Wistar rats are highly seizure susceptible at P2132 and proved to be an effective choice for this passive transfer NMDAR antibody-mediated seizure model. The CA3 region was found to have the most spontaneous epileptic activity after NMDAR antibody ICV injection, and epileptic discharges were therefore recorded from this location in vivo. The CA3 hippocampal region is also the seizure-onset zone in other murine models of epilepsy34. Another study using NMDAR antibody positive CSF recorded a higher incidence of CA3 epileptiform afterpotentials in brain slices from Wistar rats injected with NMDAR antibodies compared to controls35; these epileptiform events were not seen in other hippocampal areas37. Furthermore, electrophysiology studies with leucine-rich-glioma-1 autoantibodies (LGI1 antibodies), that cause facio-brachial dystonic seziures and limbic encephalitis, enhanced CA3 pyramidal cell excitability when applied in vitro39, and patients show focal CA3 subfield atrophy on 7T MRI following LGI1 antibody-mediated disease40. Together, these reports and our results confirm that the CA3 subfield is an important epileptogenic hippocampal region in immune-mediated types of epilepsy, justifying its further investigation.The genetic background of rodent models can confer different levels of seizure susceptibility41. Furthermore, many effective anti-seizure drugs aim to restore this balance by increasing levels of inhibition or reducing neuronal excitation42. Hypofunction in both excitatory AMPAR and NMDA receptors have been reported in epilepsies and seizure models, including antibody-induced loss of AMPARs44. Similarly, recent work in a rat model of chronic temporal lobe epilepsy (TLE) demonstrates that dysfunctional regulation and resultant hypofunction of GluA (AMPAR) subunits is an important factor in epileptogenesis45. Defects in another AMPAR subunit, GluA2, are also found in neurodevelopmental disorders such as epileptic encephalopathy in children46. Previous experiments have also demonstrated that the weakening of excitatory synapses has a paradoxical effect on neural networks, with one early in vitro and computational study showing use of AMPAR blockers (CNQX) at moderate concentrations evoked seizure-like activity47 and, more recently, AMPAR antibodies being demonstrated to produce weakened excitatory and inhibitory transimission and increased intrinsic excitability48 similar to the effects demonstrated here with NMDAR antibodies. In fact, early experiments showed epileptic seizures can be sustained even when measured extracellular calcium reached levels that would completely block chemical synaptic transmission49 highlighting the role of nonsynaptic neural network properties in epileptogenesis. Finally, as the Turrigiano laboratory has shown, synaptic scaling of excitatory amino acid receptors is co-regulated alongside intrinsic excitability48 through the actions of CaMKIV, but GABA inhibition is separately regulated, emphasising the interdependence of synaptic, cellular and circuit mechanisms in control of network excitability50.Almost all healthy brains can produce epileptic seizures in the context of acute excitation-inhibition imbalance, such as acute GABA-blockade caused by chemoconvulsants51. Loss of function mutations in both the GluN1 and GluN2A subunits of the NMDAR have been identified in patients with epilepsy and neurodevelopmental disorders53. A recent dynamic causal modelling study of NMDAR antibody encephalitis patient EEGs also showed that the deficit in signalling at NMDARs in patients with NMDAR antibody encephalitis was predominantly located at the NMDA receptors of excitatory neurons rather than at inhibitory interneurons54, as shown here in our model. By combining detailed multiscale electrophysiology and computational modelling, we provide further evidence that the direct synaptic effects of NMDAR antibody identified from whole-cell patch clamp recordings offer a quantitative explanation for the epileptic brain dynamics captured in vivo using local field potentials.That NMDAR hypofunction is proconvulsant has been shown though a variety of approaches. Genetically modified mice lacking NMDARs in CA3 pyramidal neurons were more susceptible to kainate-induced seizures, and pharmacological blockade of CA3 NMDAR in adult wild-type mice produced similar resultsincreased pyramidal cell excitability55, potentially driven by a release from inhibition by reducing putative NMDAR-mediated excitatory driving of inhibitory neuron activity, or by changing phase relationships between interacting neuronal populations. Similarly, hippocampal CA3 NMDA receptors may normally act to suppress the excitability of the recurrent network by restricting synchronous firing of CA3 neurons51, with NMDAR-blockade releasing them from this suppression. Our modelling also suggests that NMDAR antibody hypofunction may have stronger impacts on specific compartments of the affected microcircuitry.In our circuit models of NMDAR antibody-induced epileptogenesis, the parameter changes required to optimally model the associated LFP changes required not only changes in NMDAR-transmission related parameters, but also more widespread changes involving both excitatory and inhibitory neuronal populations. This would appear to fit with the co-dependent changes seen in our rodent model and those of others as reported above. In addition to such compensatory and maladaptive network changes, heterogeneity of NMDAR distribution at normal baseline function may also account for the unexpected effect of NMDAR-hypofunction in neuronal circuit dynamics. Selective forebrain pyramidal neuron NR1 knockout mice demonstrated both socio-cognitive deficits and Lastly, the integrated brain (micro-)circuits under investigation here show complex emergent dynamics arising from a range of often nonlinear interactions. This complexity can often give rise to unpredictable results, even where simple systems show a clear mapping between excitation/inhibition balance and seizure propensity. Our modelling suggests that the effective synaptic connectivity changes associated with NMDAR antibodies sensitise the circuit to perturbations in another (largely unrelated) set of parameters. This insight implies that whilst the seizure transition may be indicative of a change in a balance of parameters associated with excitation and inhibition, there may be territories of parameter space where this change is tolerated without any notable impact on the local field potential and other territories where the same change of excitation/inhibition parameter balance would produce large changes consistent with an epileptic seizure. The simulations here indicate that the NMDAR antibody models are much more sensitive to ‘ictogenesis’-associated parameter changes, suggesting that the combined parameter changes induced by NMDAR antibody have pushed the microcircuit into a territory in parameter space, where it is particularly sensitive to further perturbations.56, and modulate brain excitability primarily by interacting with receptors and ion channels on the membrane surface rather than within cells57. Pregnenolone (precursor to PregS) elevates levels of allopregnanolone and pregnenolone sulphate, a positive NMDAR receptor modulator. Pregnenolone has been used as an effective adjunctive therapy in treatment trials for schizophrenia, a psychiatric condition that shares the pathophysiology of NMDAR hypofunction60. These trials demonstrate both the safety and efficacy of Pregnenolone in humans. In vivo, pregnenolone was found to reduce the hyperlocomotion, stereotypic bouts and prepulse inhibition deficits in a mouse model of schizophrenia acutely, and improved impaired cognitive deficits with chronic treatment61. PregS, a sulfated ester of pregnenolone, is a positive NMDAR modulator63 and studies show that the mechanism of action is to increase surface expression of functional NMDARs containing the NMDAR subunit subtype 2A (NR2A) or subtype 2B (NR2B), both the most abundant subunits found in the brain; no evidence points to changes in NR2C or NR2D subunits28. In a previous study, this membrane delimited effect postulated to be via a non-canonical, G-protein and calcium-dependent mechanism occurred within 10 min of application to cortical neurons, increasing surface levels by 60–100%28; this speed of action of PregS was reproducible in vitro in our brain slice recordings. In addition to the effect of PregS on NMDARs, other CNS targets have been described, including the GABAA receptor, glycine receptor, and more recently the TRPM3 channel64 and Kir2.3 channel65. The effects of PregS on the GABAAR most commonly described are at lower, nanomolar range doses67. We found that the 100 µM dose of PregS had no effect on GABAergic neurotransmission in the brain slices from the NMDAR antibody model. A proconvulsant effect was seen in the control antibody slices, but the reverse was seen in the NMDAR antibody infused brain slices, suggesting that it was the increase of NMDARs that led to the reduction in the frequency of epileptic events.Neurosteroids are derived from cholesterol and are precursors to gonadal steroid hormones and the adrenal corticosteroids. They are produced de novo by glial cells and principal neurons, hence the term ‘neurosteroid’11. To our knowledge this is the first study to show that symptoms established for more than 48 h , can be treated with the use of the neurosteroid PregS. Whilst PregS was chosen here specifically to treat the known underlying pathophysiology, its role for the treatment of epilepsy may extend into other conditions or scenarios where the treatments that accord to the notion of reestablishing excitation-inhibition balance have failed to be effective (e.g. refractory status epilepticus)68.Previous studies with NMDAR modulators and other treatments have focused on the prevention of pathogenic antibody effects69. A single CA3 pyramidal cell can make synapses with 30,000–60,000 neurons in the ipsilateral hippocampus70. It, therefore, remains a possibility that NMDAR hypofunction affecting GABAergic interneuronal populations produces a disinhibited CA3 network72 and investigating this possibility should be a goal of further electrophysiological studies.We did not record directly from inhibitory interneurons but it is likely that both cell types are involved in driving the epileptogenesis. The CA3 network is highly complex, with CA3 pyramidal cells having convergent and divergent connections with several interneuronal types in different hippocampal regions35. Mossy fibre synapses display a markedly lower proportion of GluN2B-containing NMDARs73 than associative or commissural synapses, therefore a loss of NMDARs in this critical population of cells could also be devastating. PregS offers an attractive treatment strategy to increase NR1 and NR2B subunit NMDARs28 that could potentially mitigate the short and long-term cognitive effects seen in patients, as well as the seizures. Further detailed molecular studies will need to be done to characterize the NMDAR and subunit changes.Another limitation to this study was that the cognitive effects of NMDAR antibodies were not investigated in the animals or electrophysiologically through long-term potentiation (LTP) recordings. However, LTP recordings will be difficult to interpret with spontaneous ictal spikes as seen in these brain slices. It is known that in CA3, NMDAR-dependent  and NMDAR-independent (Mossy-fibre) forms of LTP are expressed in adjacent synapses. LTP has been shown to be reduced by NMDAR antibody positive CSF (experiments performed in Wistar rats), and this reduction may contribute to the cognitive deficits seen clinically60. There is precedence for the use of neurosteroids in other forms of epilepsy, including genetic epilepsies  as well as refractory status epilepticus74. In genetic epilepsy, research efforts have focused for some time on modifying the specific underlying genetic effect with new and repurposed drugs75. In this study, we show that there is also a possibility of providing receptor-specific neurosteroid treatment to affected patents with immune-mediated epilepsy and encephalitis.Future studies also need to focus on ascertaining the optimal brain concentrations of the neurosteroid PregS in vivo in the NMDAR antibody mediated disease models, and on determining whether these can be achieved through direct administration of PregS (e.g. by subcutaneous injection) or the precursor Pregnenolone 18. In brief, plasma was diluted 1:1 with Hartmann’s solution and passed through Protein G sepharose column beads. To elute IgG, 0.1 M glycine solution (pH 2.3) was used and the sample then neutralized with 1 M Tris pH 8. A Coomassie Plus assay kit  was used to determine protein concentrations of eluted fractions. Peak protein fractions were pooled and dialysed against Hartmann’s solution, and IgG concentrated using Amicon ultra-4 10KDa filter units, and then filter-sterilized. Nanodrop 3300  was used to determine final IgG concentration (37 mg/ml). In addition, two different recombinant NMDAR antibody specific human monoclonal antibodies were donated for use; SSM5 (4.3 mg/ml)8 and 003-102 (3.4 mg/ml)3. Both of these NMDAR antibody monoclonal antibodies have previously been shown to have in vitro and in vivo pathogenic effects8.Three human-derived NMDAR antibody preparations were obtained for experimental use. The first was NMDAR antibody positive IgG prepared from plasma obtained, with informed consent at plasmapheresis from a patient , using Protein G beads3, and 12D7 (6 mg/ml)8.Control antibodies (Control antibodies) were human derived IgG from one healthy individual , and two non-brain reactive monoclonal Control antibodies; mGO53 (514.5 µg/ml)As all antibody preparations had previously been used in the studies specified and were known to be pathogenic, and stocks for some preparations were limited, a direct comparison was not made between each antibody preparation of the epileptogenic effects. The three NMDAR antibody preparations will be collectively referred to as “NMDAR antibodies” throughout the paper and the three control preparations as “Control antibodies”. Detailed description of antibody use per experiment is provided in Supplementary Table 45, we hypothesized that the use of juvenile male Wistar rats would increase the probability of observing spontaneous seizures in a NMDAR antibody passive transfer model. Thirty-one post-natal day 21 (P21) male Wistar rats, weighing 50–58 g, were used for the in vitro experiments and 12 were used for the in vivo experiments. The animals were housed in temperature- and humidity-controlled conditions with a 12 h/12 h light/dark cycle, and allowed to feed and drink ad libitum. All procedures were compliant with current UK Home Office guidelines as required by the Home Office Animals (Scientific Procedures) Act 1986. They were carried out under the authority and procedural approval of a UK Home Office approved project license and in line with ARRIVE guidelines. Local ethical approval for the study was granted by the Aston Bioethics Committee, University of Aston, Birmingham, UK.Given our experience in other rodent epilepsy models18. A 0.5 cm incision was made caudally over the skull surface which was then exposed before a single burr hole was made for ICV injection. The co-ordinates for the left lateral ventricle were 1.5 mm left lateral and 0.6 mm caudal from bregma76. 10 micrograms (10 mcg) of SSM5 , or non-reactive control antibody (12D7) or 8 μL of NMDAR antibody IgG18 or healthy control IgG was used. Antibody preparations were coded to allow for blinded analysis. The antibody was injected over twenty minutes using a Hamilton syringe with 33 gauge needle to a depth of 3.2 mm from skull surface. Following injection, the syringe was left in place for ten minutes before being withdrawn over twenty minutes. The skin was sutured and animal allowed to recover before being replaced in home cage.ICV injection was performed under isoflurane anaesthesia with rats fixed in a stereotaxic frame77. The slices were transferred to a storage chamber containing standard aCSF  and constantly bubbled with carbogen (95% O2/5% CO2) at room-temperature for at least one hour prior to recording. Slices were then transferred to an interface chamber  and continuously perfused with aCSF (2–3 ml/min) maintained at 30–31 °C and visualised with a SZ51 stereomicroscope (Olympus). A Flaming-Brown micropipette puller  was used to pull borosilicate glass microelectrodes with an open tip resistance of 1–3 MΩ when filled with aCSF. Electrodes were inserted into CA3 and CA1 regions of the hippocampus using Narishige MM-3  micromanipulators and signals were recorded using an EXT-02 headstage and amplifier (NPI Electronic). HumBugs (Quest Scientific) were used to remove electrical 50 Hz interference from recordings. The signal was then amplified ×100 via an EX10-2F amplifier (NPI Electronics) and filtered  and further amplified ×10 using a LHBF-48X amplifier (NPI Electronics). Signals were then digitised at 10 KHz using an analogue to digital converter . LFP recordings were assessed using Spike2 software (CED) for spontaneous epileptiform activity. Spike2 was used to calculate the root mean square (RMS) amplitude of each recording. Epileptiform activity was classified as an event when it displayed an amplitude greater than four-fold the RMS amplitude, providing the event count, while the time difference between these events provided the interevent interval. A custom MATLAB script was used to prevent false event detection, removing those with an interevent interval less than 15 ms. Statistical analysis was conducted using Graphpad Prism 8 (GraphPad Software Inc), statistical tests used to compare groups (Mann-Whitney) were one-tailed. Measurements were expressed as mean (M) ± standard error of the mean (SEM).Forty-eight hours after ICV injection or at the end of the recording period in telemetry experiments, rats were anaesthetized using isoflurane followed, after loss of consciousness, by pentobarbital  and xylazine . Transcardial perfusion was then performed using ice-cold artificial cerebrospinal fluid (cutting aCSF) in which NaCl had been replaced with equimolar sucrose. Animals were decapitated, and the brain was removed and placed in ice-cold cutting aCSF. Sagittal slices at 450μm were cut using a vibratome 2, 10 TEA-Cl, 4 ATP-Na, 0.3 GTP-Na and 1 IEM 1460 (titrated with CsOH to pH 7.25) at 290–295 mOsm for IPSCs. For EPSCs the internal solution contained (in mM): 40 HEPES, 1 Qx-314, 0.6 EGTA, 2 NaCl, 5 Mg-gluconate, 5 TEA-Cl, 10 Phospho-Creatinine, 4 ATP-Na, 0.3 GTP-Na (titrated with CsOH to pH 7.3) at 285 mOsm for EPSCs. The EPSCs and IPSCs were recorded as apparent inward currents at −70 mV using an Axopatch 200B amplifier . Signals were low-pass filtered at 5 kHz with an 8-pole Bessel filter and digitised at 10 kHz using a Digidata 1440 A and pClamp software (Molecular Devices). For intrinsic cell excitability experiments, whole-cell current-clamp recordings were made from hippocampal neurons using an Axopatch 700 series amplifier  and with a K-gluconate-based intracellular solution containing (in mM) K-gluconate (120) hepes (40) KCL (10) Na2ATP (2) Na-GTP (0.3) MgCl2 (3) EGTA (0.5). Membrane potential was manually adjusted when required to −60 mV by continuous somatic current injection. Membrane response and action potential firing were measured by applying depolarizing steps of current (duration of 1 ms at 10pA increments). For spike/current intensity curves, the number of action potentials was plotted against the injected current. Input resistance was measured from the slope of a linear regression fit to the voltage–current plotted graph between −80 pA to 0 pA. Data were analysed using Axograph and Prism 8, statistical tests used to compare groups (Mann–Whitney and Wilcoxon matched pairs ranked tests) were one-tailed. Measurements are expressed as mean median ± SEM.For whole-cell patch clamp recordings, brain slices were prepared as above but sliced at a thickness of 350 µm. For recording, the slices were transferred to a submerged chamber  and visualised using Nomarski infra-red optics on a BX51WI Microscope (Olympus). A Flaming-Brown micropipette puller  was used to pull borosilicate glass microelectrodes of 4–6 MΩ for recordings. For voltage clamp recordings, electrodes were filled with an internal solution containing (in mM): 100 CsCl, 40 HEPES, 1 Qx-314, 0.6 EGTA, 5 MgCl79. Briefly, human tissue was obtained with informed parental or guardian consent from paediatric patients prior to them undergoing epilepsy surgery at Birmingham Children’s Hospital. Ethical approval was obtained from the Black Country Local Ethics Committee , and from Aston University’s ethics committee (Project 308 cellular studies in epilepsy) and through the Research and Development Department at Birmingham Children’s Hospital (IRAS ID12287). Brain tissue was obtained from 7 patients (F:M 4:3), median age 6 years (range 4–16 years). Surgical procedures included frontal resection (three), temporal parietal occipital disconnection (one), temporal lobectomy (two), and right hemispherectomy (one) as described in Supplementary Table 79.For human tissue recordings, brain slices were prepared as above using choline-based artificial cerebrospinal fluidFor all in vitro electrophysiology experiments, Pregnenolone sulphate  was prepared as 1 M stock using dimethyl sulfoxide (DMSO). A concentration of 100 µM was used for all electrophysiology experiments. MK-801  was prepared with distilled water as 10 mM stock and used at 10 µM concentrations.Following in vitro electrophysiology experiments, brain slices were briefly fixed in 4% paraformaldehyde (PFA) for 45 min. To determine NMDAR antibody or control antibody binding, the sections were rinsed with phosphate-buffered saline (PBS) and then incubated in anti-human IgG Alexa-Fluor-488 at 1 in 1000  overnight at 4 °C. Sections were washed and mounted with aqueous mounting medium containing DAPI. Images of hippocampal sections were taken on a Tandem Confocal Scanning SP5 II microscope (Leica Microsystems Ltd) using a (10×/0.30) dry objective lens. Fluorescence was excited with a 488 nm argon laser at 23% power (emission bandwidth 504nm-564nm). Confocal micrographs were acquired at 1024 pixels2 with actual area of 1.48mm2, and scanning speed 100 Hz.Biocytin  was added to the electrode filling solution at a concentration of 5 mg/ml to determine the characteristics of the recorded cell during whole-cell patch clamp experiments. Neurons were filled with biocytin and slices were fixed overnight at 4 °C in 100 mM PBS (pH 7.3) containing 4% PFA . After washing in PBS, slices were incubated for 18 h in PBS supplemented with 1% Triton X-100 and 0.2% streptavidin Alexa Fluor 488 conjugate (Invitrogen) at 4–6 °C. Sections were washed and mounted with aqueous mounting medium containing DAPI for imaging.50 ratio of hippocampal sections was determined using 5 regions of interest (ROI) of the same size from the molecular outer layer and from the granular cell layer of CA3. The mean fluorescent intensity Log EC50 value of the outer molecular layer was normalised by dividing it by the mean fluorescent intensity Log EC50 of nonspecific staining in the granular cell layer. This process provided a ratio for each replicate. All images were analysed through FIJI by converting them to grayscale (8-bit) before generating cumulative pixel intensity histograms for each ROI which were calculated using a customized macro programme18. Any statistical analysis performed was conducted in GraphPad Prism 8.Fluorescent intensity Log EC8. Two osmotic pumps per animal were prepared by loading with monoclonal antibodies or human IgG and then connected to polyethylene tubing 69 mm-×-1.14 mm diameter  and a double osmotic pump connector intraventricular cannula . Pumps were left overnight in sterile saline solution at 37 °C. Rats were placed in a stereotaxic frame for surgery performed under isoflurane anaesthesia. Osmotic pumps were placed subcutaneously and attached cannulae inserted into both lateral ventricles . The EEG transmitter was placed in a subcutaneous pocket formed over the right flank with a single skin incision and blunt tissue dissection ), and depth electrode (W-Electrode (SCE-W), OSI) placed in the left hippocampus  with a reference electrode implanted on the contralateral skull surface . The cannula and skull electrodes were secured with dental cement25.Osmotic pumps  were used for cerebroventricular infusion of monoclonal antibodies and human IgG 80. In brief, for automated ictal event detection, video-EEG matching was used to identify ictal EEG events. The Event Classifier (OSI) was then used to classify one-second segments of EEG according to program metrics  enabling similar events to cluster together when plotted. This generated a library of ictal events that allowed fast identification of abnormal EEG events by automated comparison to the library (http://www.opensourceinstruments.com/Electronics/A3018/Seizure_Detection.html). Powerband analysis was carried out using a custom-designed macro. Statistical analysis was conducted using Graphpad Prism 8 (GraphPad Software Inc.).EEG data (wide band pass 0.2–160 Hz sampled at 512 samples per second) was collected and recorded from freely moving animals placed in a custom-built Faraday cage with an aerial. Transmitter signals were continuously recorded using Neuroarchiver software (OSI) and analysed as previously describedData in each graph was analysed with two-way ANOVA and post-hoc test, Mann–Whitney or Wilcoxon paired rank test, specified in figure legend. In in vitro electrophysiology experiments, individual points represent brain slices or cells recorded from at least three different animals or human brain tissue samples per experimental group. In in vivo experiments, data were collected from six animals in each group. The reproducibility of the data were maintained by using different NMDAR-Ab preparations. All graphs are expressed a mean (M) with bars representing standard error of the mean (SEM).https://www.fil.ion.ucl.ac.uk/spm/). All analysis code and raw data are available online at 10.17605/OSF.IO/GUPBF81. Analogous to previous work82, we modelled LFP spectra recorded in rat hippocampal tissue as arising from a single canonical microcircuit at steady state83. This ‘canonical microcircuit’ is a layered set of recurrently coupled excitatory and inhibitory neural masses86. Originally conceived as a description of layered cortical population activity, the canonical microcircuit model has also been shown to offer a parsimonious explanation of rodent hippocampal activity in different physiological and abnormal states87. The four populations of the canonical microcircuit act as two coupled oscillator pairs: One superficial oscillator typically characterised by fast (beta/gamma-range) frequencies and consisting of ‘superficial pyramidal cells’ and ‘spiny stellate cells’; and one deep oscillator with typically slow  frequencies consisting of inhibitory interneurons and deep pyramidal cells. This model is a description of neuronal interactions at the population level. Effective synaptic connectivity is parameterized by three key parameters: effective coupling strength (γ), a postsynaptic time constant (τ), and the slope of a parameterised sigmoid function (σ) that encodes population response variance88. The free neuronal parameters in the model implemented here are summarized in Table Dynamic causal modelling (DCM) was performed using the academic software package SPM12  and empirical likelihoods (‘the data’) to generate posterior densities (‘parameter estimates’)91.DCM employs a standard variational Laplace model inversion to (i) estimate posterior densities of model parameters given the data and some prior values, and (ii) provide an estimate for the Bayesian model evidence using the free energy approximationThe estimation of the Bayesian model evidence allowed us to compare evidence for models of the same data under different assumptions encoded in different priors. Specifically, we wished to test the hypothesis that NMDAR antibody-induced changes measured using whole-cell patch clamp recordings contribute to the LFP features observed in vivo and that were modelled with the DCM. To test this hypothesis, we, therefore, performed several DCM inversions on the same data under quantitatively different priors, and compared the model evidence for each of these models.Specifically we followed the following procedure; we first wanted to identify which synaptic parameters we would expect to be altered in the NMDAR antibody in vivo experiments, based on the microscale in vitro recordings. To be able to make quantitative predictions about such parameters we quantified relevant features of synaptic transmission in whole-cell patch clamp recordings. Using these features, we generated quantitative predictions of synaptic parameter differences between control antibody and NMDAR antibody conditions. To be able to infer synaptic coupling parameters underlying the local field potential data, we extracted data from different relevant conditions and states  to then fit the microcircuit models using DCM to the average power spectral densities of each of these conditions. We next wished to identify a synaptic parameter combination that best describes a physiological, interictal state from which all other conditions emerge by changes in synaptic coupling. We, therefore, fitted a single canonical microcircuit model to control antibody data, using the inferred parameters as priors in subsequent Bayesian model inversions in the next steps. To address the question whether the observed microscale changes in synaptic function contribute to the observed LFP features, we fitted a single canonical microcircuit to interictal NMDAR antibody data under different sets of priors—including those that are informed by microscale data, and those that are not. This allowed us to perform Bayesian model comparison between these models. Here we would expect models that contain microscale-derived prior information to outperform other models if the modelled microscale features contribute to the LFP data. To address the question which synaptic mechanisms best explain condition- and state-differences, we fitted a single canonical microcircuit to seizure NMDAR antibody data, then quantified the parameter differences between different DCM models. The objective here was to quantify the main differences between conditions (control antibody vs NMDAR antibody) and states  in terms of neuronal population model parameters.Further information on research design is available in the Transparent Peer Review FileDescription of Supplementary FilesSupplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Data 1Reporting Summary