Altered synaptic connectivity in an in vitro human model of STXBP1 encephalopathy.

Early infantile developmental and epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans, at the relevant time period in utero. To address this, we established an in vitro assay to study the impact of gene variants on the developing human brain by using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14-17 post-conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically and form new synaptic networks. This preparation thus permits the study of genetic manipulations and their downstream effects on an intact developing human cortical network. We focused on STXBP1 haploinsufficiency, which is among the most common genetic causes of developmental and epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a reduced density of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

Mouse genetics reveals Barttin as a genetic modifier of Joubert syndrome.

Genetic and phenotypic heterogeneity and the lack of sufficiently large patient cohorts pose a significant challenge to understanding genetic associations in rare disease. Here we identify Bsnd (alias Barttin) as a genetic modifier of cystic kidney disease in Joubert syndrome, using a Cep290-deficient mouse model to recapitulate the phenotypic variability observed in patients by mixing genetic backgrounds in a controlled manner and performing genome-wide analysis of these mice. Experimental down-regulation of Bsnd in the parental mouse strain phenocopied the severe cystic kidney phenotype. A common polymorphism within human BSND significantly associates with kidney disease severity in a patient cohort with CEP290 mutations. The striking phenotypic modifications we describe are a timely reminder of the value of mouse models and highlight the significant contribution of genetic background. Furthermore, if appropriately managed, this can be exploited as a powerful tool to elucidate mechanisms underlying human disease heterogeneity.

Tyramide signal amplification coupled with multiple immunolabeling and RNAScope in situ hybridization in formaldehyde-fixed paraffin-embedded human fetal brain.

Several strategies have been recently introduced to improve the practicality of multiple immunolabeling and RNA in situ hybridization protocols. Tyramide signal amplification (TSA) is a powerful method used to improve the detection sensitivity of immunohistochemistry. RNAScope is a novel commercially available in situ hybridization assay for the detection of RNA expression. In this work, we describe the use of TSA and RNAScope in situ hybridization as extremely sensitive and specific methods for the evaluation of protein and RNA expression in formaldehyde-fixed paraffin-embedded human fetal brain sections. These two techniques, when properly optimized, were highly compatible with routine formaldehyde-fixed paraffin-embedded tissue that preserves the best morphological characteristics of delicate fetal brain samples, enabling an unparalleled ability to simultaneously visualize the expression of multiple protein and mRNA of genes that are sparsely expressed in the human fetal telencephalon.

Creative Destruction: A Basic Computational Model of Cortical Layer Formation.

One of the most characteristic properties of many vertebrate neural systems is the layered organization of different cell types. This cytoarchitecture exists in the cortex, the retina, the hippocampus, and many other parts of the central nervous system. The developmental mechanisms of neural layer formation have been subject to substantial experimental efforts. Here, we provide a general computational model for cortical layer formation in 3D physical space. We show that this multiscale, agent-based model, comprising two distinct stages of apoptosis, can account for the wide range of neuronal numbers encountered in different cortical areas and species. Our results demonstrate the phenotypic richness of a basic state diagram structure. Importantly, apoptosis allows for changing the thickness of one layer without automatically affecting other layers. Therefore, apoptosis increases the flexibility for evolutionary change in layer architecture. Notably, slightly changed gene regulatory dynamics recapitulate the characteristic properties observed in neurodevelopmental diseases. Overall, we propose a novel computational model using gene-type rules, exhibiting many characteristics of normal and pathological cortical development.

Hippocampal network hyperexcitability in young transgenic mice expressing human mutant alpha-synuclein.

Abnormal excitability in cortical networks has been reported in patients and animal models of Alzheimer's disease (AD), and other neurodegenerative conditions. Whether hyperexcitability is a core feature of alpha(α)-synucleinopathies, including dementia with Lewy bodies (DLB) is unclear. To assess this, we used two murine models of DLB that express either human mutant α-synuclein (α-syn) the hA30P, or human wild-type α-syn (hWT-α-syn) mice. We observed network hyperexcitability in vitro in young (2-5 months), pre-symptomatic transgenic α-syn mice. Interictal discharges (IIDs) were seen in the extracellular local field potential (LFP) in the hippocampus in hA30P and hWT-α-syn mice following kainate application, while only gamma frequency oscillations occurred in control mice. In addition, the concentration of the GABAA receptor antagonist (gabazine) needed to evoke IIDs was lower in slices from hA30P mice compared to control mice. hA30P mice also showed increased locomotor activity in the open field test compared to control mice. Intracellular recordings from CA3 pyramidal cells showed a more depolarised resting membrane potential in hA30P mice. Quadruple immunohistochemistry for human α-syn, and the mitochondrial markers, porin and the complex IV enzyme cytochrome c oxidase subunit 1 (COX1) in parvalbumin (PV+)-expressing interneurons showed that 25% of PV+ cells contained human α-syn in hA30P mice. While there was no change in PV expression, COX1 expression was significantly increased in PV+ cells in hA30P mice, perhaps reflecting a compensatory change to support PV+ interneuron activity. Our findings suggest that hippocampal network hyperexcitability may be an important early consequence of α-syn-mediated impairment of neuronal/synaptic function, which occurs without any overt loss of PV interneurons. The therapeutic benefit of targeting network excitability early in the disease stage should be explored with respect to α-synucleinopathies such as DLB.

Charting the protomap of the human telencephalon.

The cerebral cortex is divided stereotypically into a number of functionally distinct areas. According to the protomap hypothesis formulated by Rakic neural progenitors in the ventricular zone form a mosaic of proliferative units that provide a primordial species-specific cortical map. Positional information of newborn neurons is maintained during their migration to the overlying cortical plate. Much evidence has been found to support this hypothesis from studies of primary cortical areas in mouse models in particular. Differential expansion of cortical areas and the introduction of new functional modules during evolution might be the result of changes in the progenitor cells. The human cerebral cortex shows a wide divergence from the mouse containing a much higher proportion of association cortex and a more complicated regionalised repertoire of neuron sub-types. To what extent does the protomap hypothesis hold true for the primate brain? This review summarises a growing number of studies exploring arealised gene expression in the early developing human telencephalon. The evidence so far is that the human and mouse brain do share fundamental mechanisms of areal specification, however there are subtle differences which could lead us to a better understanding of cortical evolution and the origins of neurodevelopmental diseases.

Thalamocortical Afferents Innervate the Cortical Subplate much Earlier in Development in Primate than in Rodent.

The current model, based on rodent data, proposes that thalamocortical afferents (TCA) innervate the subplate towards the end of cortical neurogenesis. This implies that the laminar identity of cortical neurons is specified by intrinsic instructions rather than information of thalamic origin. In order to determine whether this mechanism is conserved in the primates, we examined the growth of thalamocortical (TCA) and corticofugal afferents in early human and monkey fetal development. In the human, TCA, identified by secretagogin, calbindin, and ROBO1 immunoreactivity, were observed in the internal capsule of the ventral telencephalon as early as 7-7.5 PCW, crossing the pallial/subpallial boundary (PSB) by 8 PCW before the calretinin immunoreactive corticofugal fibers do. Furthermore, TCA were observed to be passing through the intermediate zone and innervating the presubplate of the dorsolateral cortex, and already by 10-12 PCW TCAs were occupying much of the cortex. Observations at equivalent stages in the marmoset confirmed that this pattern is conserved across primates. Therefore, our results demonstrate that in primates, TCAs innervate the cortical presubplate at earlier stages than previously demonstrated by acetylcholinesterase histochemistry, suggesting that pioneer thalamic afferents may contribute to early cortical circuitry that can participate in defining cortical neuron phenotypes.

Expression of ventral telencephalon transcription factors ASCL1 and DLX2 in the early fetal human cerebral cortex.

In rodent ventral telencephalon, diffusible morphogens induce expression of the proneural transcription factor ASCL1, which in turn induces expression of the transcription factor DLX2 that controls differentiation of cortical interneuron precursors and their tangential migration to the cerebral cortex. RNAseq analysis of human fetal samples of dorsal telencephalon revealed consistently high cortical expression of ASCL1 and increasing expression of DLX2 between 7.5 and 17 post-conceptional weeks (PCW). We explored whether cortical expression of these genes represented a population of intracortically derived interneuron precursors. Immunohistochemistry revealed an ASCL1+ /DLX2+ population of progenitor cells in the human ganglionic eminences between 6.5 and 12 PCW, but in the cortex there also existed a population of ASCL1+ /DLX2- progenitors in the subventricular zone (SVZ) that largely co-expressed cortical markers PAX6 or TBR2, although a few ASCL1+ /PAX6- progenitors were observed in the ventricular zone (VZ) and ASCL1+ cells expressing the interneuron marker GAD67 were present in the SVZ. Although rare in the VZ, DLX2+ cells progressively increased in number between 8 and 12 PCW across the cortical wall and the majority co-expressed LHX6 and originated either in the MGE, migrating to the lateral cortex, or from the septum, populating the medial wall. A minority co-expressed COUP-TFII, which identifies cells from the caudal ganglionic eminence (CGE). By 19 PCW, a significant increase in expression of DLX2 and ASCL1 was observed in the cortical VZ with a small proportion expressing both proteins. The DLX2+ cells did not co-express a cell division marker, so were not progenitors. The majority of DLX2+ cells throughout the cortical plate expressed COUP-TFII rather than LHX6+ . As the VZ declined as a proliferative zone it appeared to be re-defined as a migration pathway for COUP-TFII+ /DLX2+ interneurons from CGE to cortex. Therefore, in developing human cortex, ASCL1 expression predominantly marks a population of intermediate progenitors giving rise to glutamatergic neurons. DLX2 expression predominantly defines post-mitotic interneuron precursors.

New insights into the development of the human cerebral cortex.

The cerebral cortex constitutes more than half the volume of the human brain and is presumed to be responsible for the neuronal computations underlying complex phenomena, such as perception, thought, language, attention, episodic memory and voluntary movement. Rodent models are extremely valuable for the investigation of brain development, but cannot provide insight into aspects that are unique or highly derived in humans. Many human psychiatric and neurological conditions have developmental origins but cannot be studied adequately in animal models. The human cerebral cortex has some unique genetic, molecular, cellular and anatomical features, which need to be further explored. The Anatomical Society devoted its summer meeting to the topic of Human Brain Development in June 2018 to tackle these important issues. The meeting was organized by Gavin Clowry (Newcastle University) and Zoltán Molnár (University of Oxford), and held at St John's College, Oxford. The participants provided a broad overview of the structure of the human brain in the context of scaling relationships across the brains of mammals, conserved principles and recent changes in the human lineage. Speakers considered how neuronal progenitors diversified in human to generate an increasing variety of cortical neurons. The formation of the earliest cortical circuits of the earliest generated neurons in the subplate was discussed together with their involvement in neurodevelopmental pathologies. Gene expression networks and susceptibility genes associated to neurodevelopmental diseases were discussed and compared with the networks that can be identified in organoids developed from induced pluripotent stem cells that recapitulate some aspects of in vivo development. New views were discussed on the specification of glutamatergic pyramidal and γ-aminobutyric acid (GABA)ergic interneurons. With the advancement of various in vivo imaging methods, the histopathological observations can be now linked to in vivo normal conditions and to various diseases. Our review gives a general evaluation of the exciting new developments in these areas. The human cortex has a much enlarged association cortex with greater interconnectivity of cortical areas with each other and with an expanded thalamus. The human cortex has relative enlargement of the upper layers, enhanced diversity and function of inhibitory interneurons and a highly expanded transient subplate layer during development. Here we highlight recent studies that address how these differences emerge during development focusing on diverse facets of our evolution.

Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control.

SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein-RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites.

Human neural stem cells dispersed in artificial ECM form cerebral organoids when grafted in vivo.

Human neural stem cells (hNSC) derived from induced pluripotent stem cells can be differentiated into neurons that could be used for transplantation to repair brain injury. In this study we dispersed such hNSC in a three-dimensional artificial extracellular matrix (aECM) and compared their differentiation in vitro and following grafting into the sensorimotor cortex (SMC) of postnatal day (P)14 rat pups lesioned by localised injection of endothelin-1 at P12. After 10-43 days of in vitro differentiation, a few cells remained as PAX6+ neuroprogenitors but many more resembled post-mitotic neurons expressing doublecortin, ß-tubulin and MAP2. These cells remained dispersed throughout the ECM, but with extended long processes for over 50 µm. In vivo, by 1 month post grafting, cells expressing human specific markers instead organised into cerebral organoids: columns of tightly packed PAX6 co-expressing progenitor cells arranged around small tubular lumen in rosettes, with a looser network of cells with processes around the outside co-expressing markers of immature neurons including doublecortin, and CTIP2 characteristic of corticofugal neurons. Host cells also invaded the graft including microglia, astrocytes and endothelial cells forming blood vessels. By 10 weeks post-grafting, the organoids had disappeared and the aECM had started to break down with fewer transplanted cells remaining. In vitro, cerebral organoids form in rotating incubators that force oxygen and nutrients to the centre of the structures. We have shown that cerebral organoids can form in vivo; intrinsic factors may direct their organisation including infiltration by host blood vessels.

The Transcription Factors COUP-TFI and COUP-TFII have Distinct Roles in Arealisation and GABAergic Interneuron Specification in the Early Human Fetal Telencephalon.

In human telencephalon at 8-12 postconceptional weeks, ribonucleic acid quantitative sequencing and immunohistochemistry revealed cortical chicken ovalbumin upstream promotor-transcription factor 1 (COUP-TFI) expression in a high ventro-posterior to low anterior gradient except for raised immunoreactivity in the anterior ventral pallium. Unlike in mouse, COUP-TFI and SP8 were extensively co-expressed in dorsal sensory neocortex and dorsal hippocampus whereas COUPTFI/COUPTFII co-expression defined ventral temporal cortex and ventral hippocampus. In the ganglionic eminences (GEs) COUP-TFI immunoreactivity demarcated the proliferative zones of caudal GE (CGE), dorsal medial GE (MGE), MGE/lateral GE (LGE) boundary, and ventral LGE whereas COUP-TFII was limited to ventral CGE and the MGE/LGE boundary. Co-labeling with gamma amino butyric acidergic interneuron markers revealed that COUP-TFI was expressed in subpopulations of either MGE-derived (SOX6+) or CGE-derived (calretinin+/SP8+) interneurons. COUP-TFII was mainly confined to CGE-derived interneurons. Twice as many GAD67+ cortical cells co-labeled for COUP-TFI than for COUP-TFII. A fifth of COUP-TFI cells also co-expressed COUP-TFII, and cells expressing either transcription factor followed posterior or anterio-lateral pathways into the cortex, therefore, a segregation of migration pathways according to COUP-TF expression as proposed in mouse was not observed. In cultures differentiated from isolated human cortical progenitors, many cells expressed either COUP-TF and 30% also co-expressed GABA, however no cells expressed NKX2.1. This suggests interneurons could be generated intracortically from progenitors expressing either COUP-TF.

Neurexins 1-3 Each Have a Distinct Pattern of Expression in the Early Developing Human Cerebral Cortex.

Neurexins (NRXNs) are presynaptic terminal proteins and candidate neurodevelopmental disorder susceptibility genes; mutations presumably upset synaptic stabilization and function. However, analysis of human cortical tissue samples by RNAseq and quantitative real-time PCR at 8-12 postconceptional weeks, prior to extensive synapse formation, showed expression of all three NRXNs as well as several potential binding partners. However, the levels of expression were not identical; NRXN1 increased with age and NRXN2 levels were consistently higher than for NRXN3. Immunohistochemistry for each NRXN also revealed different expression patterns at this stage of development. NRXN1 and NRXN3 immunoreactivity was generally strongest in the cortical plate and increased in the ventricular zone with age, but was weak in the synaptogenic presubplate (pSP) and marginal zone. On the other hand, NRXN2 colocalized with synaptophysin in neurites of the pSP, but especially with GAP43 and CASK in growing axons of the intermediate zone. Alternative splicing modifies the role of NRXNs and we found evidence by RNAseq for exon skipping at splice site 4 and concomitant expression of KHDBRS proteins which control this splicing. NRXN2 may play a part in early cortical synaptogenesis, but NRXNs could have diverse roles in development including axon guidance, and intercellular communication between proliferating cells and/or migrating neurons.

Distinct expression patterns for type II topoisomerases IIA and IIB in the early foetal human telencephalon.

TOP2A and TOP2B are type II topoisomerase enzymes that have important but distinct roles in DNA replication and RNA transcription. Recently, TOP2B has been implicated in the transcription of long genes in particular that play crucial roles in neural development and are susceptible to mutations contributing to neurodevelopmental conditions such as autism and schizophrenia. This study maps their expression in the early foetal human telencephalon between 9 and 12 post-conceptional weeks. TOP2A immunoreactivity was restricted to cell nuclei of the proliferative layers of the cortex and ganglionic eminences (GE), including the ventricular zone and subventricular zone (SVZ) closely matching expression of the proliferation marker KI67. Comparison with sections immunolabelled for NKX2.1, a medial GE (MGE) marker, and PAX6, a cortical progenitor cell and lateral GE (LGE) marker, revealed that TOP2A-expressing cells were more abundant in MGE than the LGE. In the cortex, TOP2B is expressed in cell nuclei in both proliferative (SVZ) and post-mitotic compartments (intermediate zone and cortical plate) as revealed by comparison with immunostaining for PAX6 and the post-mitotic neuron marker TBR1. However, co-expression with KI67 was rare. In the GE, TOP2B was also expressed by proliferative and post-mitotic compartments. In situ hybridisation studies confirmed these patterns of expression, except that TOP2A mRNA is restricted to cells in the G2/M phase of division. Thus, during early development, TOP2A is likely to have a role in cell proliferation, whereas TOP2B is expressed in post-mitotic cells and may be important in controlling expression of long genes even at this early stage.

The early fetal development of human neocortical GABAergic interneurons.

GABAergic interneurons are crucial to controlling the excitability and responsiveness of cortical circuitry. Their developmental origin may differ between rodents and human. We have demonstrated the expression of 12 GABAergic interneuron-associated genes in samples from human neocortex by quantitative rtPCR from 8 to 12 postconceptional weeks (PCW) and shown a significant anterior to posterior expression gradient, confirmed by in situ hybridization or immunohistochemistry for GAD1 and 2, DLX1, 2, and 5, ASCL1, OLIG2, and CALB2. Following cortical plate (CP) formation from 8 to 9 PCW, a proportion of cells were strongly stained for all these markers in the CP and presubplate. ASCL1 and DLX2 maintained high expression in the proliferative zones and showed extensive immunofluorescent double-labeling with the cell division marker Ki-67. CALB2-positive cells increased steadily in the SVZ/VZ from 10 PCW but were not double-labeled with Ki-67. Expression of GABAergic genes was generally higher in the dorsal pallium than in the ganglionic eminences, with lower expression in the intervening ventral pallium. It is widely accepted that the cortical proliferative zones may generate CALB2-positive interneurons from mid-gestation; we now show that the anterior neocortical proliferative layers especially may be a rich source of interneurons in the early neocortex.

The tissue-specific RNA binding protein T-STAR controls regional splicing patterns of neurexin pre-mRNAs in the brain.

The RNA binding protein T-STAR was created following a gene triplication 520-610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain.

An enhanced role and expanded developmental origins for gamma-aminobutyric acidergic interneurons in the human cerebral cortex.

Human beings have considerably expanded cognitive abilities compared with all other species and they also have a relatively larger cerebral cortex compared with their body size. But is a bigger brain the only reason for higher cognition or have other features evolved in parallel? Humans have more and different types of GABAergic interneurons, found in different places, than our model species. Studies are beginning to show differences in function. Is this expanded repertoire of functional types matched by an evolution of their developmental origins? Recent studies support the idea that generation of interneurons in the ventral telencephalon may be more complicated in primates, which have evolved a large and complex outer subventricular zone in the ganglionic eminences. In addition, proportionally more interneurons appear to be produced in the caudal ganglionic eminence, the majority of which populate the superficial layers of the cortex. Whether or not the cortical proliferative zones are a source of interneurogenesis, and to what extent and of what significance, is a contentious issue. As there is growing evidence that conditions such as autism, schizophrenia and congenital epilepsy may have developmental origins in the failure of interneuron production and migration, it is important we understand fully the similarities and differences between human development and our animal models.

Gap junction networks can generate both ripple-like and fast ripple-like oscillations.

Fast ripples (FRs) are network oscillations, defined variously as having frequencies of > 150 to > 250 Hz, with a controversial mechanism. FRs appear to indicate a propensity of cortical tissue to originate seizures. Here, we demonstrate field oscillations, at up to 400 Hz, in spontaneously epileptic human cortical tissue in vitro, and present a network model that could explain FRs themselves, and their relation to 'ordinary' (slower) ripples. We performed network simulations with model pyramidal neurons, having axons electrically coupled. Ripples (< 250 Hz) were favored when conduction of action potentials, axon to axon, was reliable. Whereas ripple population activity was periodic, firing of individual axons varied in relative phase. A switch from ripples to FRs took place when an ectopic spike occurred in a cell coupled to another cell, itself multiply coupled to others. Propagation could then start in one direction only, a condition suitable for re-entry. The resulting oscillations were > 250 Hz, were sustained or interrupted, and had little jitter in the firing of individual axons. The form of model FR was similar to spontaneously occurring FRs in excised human epileptic tissue. In vitro, FRs were suppressed by a gap junction blocker. Our data suggest that a given network can produce ripples, FRs, or both, via gap junctions, and that FRs are favored by clusters of axonal gap junctions. If axonal gap junctions indeed occur in epileptic tissue, and are mediated by connexin 26 (recently shown to mediate coupling between immature neocortical pyramidal cells), then this prediction is testable.