Publications

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from patterns of spatiotemporal neuronal activity. These dynamic patterns emerging from functional groups of interconnected neuronal ensembles facilitate precise computations for processing and coding multiscale neural information, thereby driving higher brain functions. To probe the computational principles of neural dynamics underlying this complexity and investigate the multiscale impact of biological processes in health and disease, large-scale simultaneous recordings have become instrumental. Here, a high-density microelectrode array (HD-MEA) is employed to study two modalities of neural dynamics – hippocampal and olfactory bulb circuits from ex-vivo mouse brain slices and neuronal networks from in-vitro cell cultures of human induced pluripotent stem cells (iPSCs). The HD-MEA platform, with 4096 microelectrodes, enables non-invasive, multi-site, label-free recordings of extracellular firing patterns from thousands of neuronal ensembles simultaneously at high spatiotemporal resolution. This approach allows the characterization of several electrophysiological network-wide features, including single/-multi-unit spiking activity patterns and local field potential oscillations. To scrutinize these multidimensional neural data, we have developed several computational tools incorporating machine learning algorithms, automatic event detection and classification, graph theory, and other advanced analyses. By supplementing these computational pipelines with this platform, we provide a methodology for studying the large, multiscale, and multimodal dynamics from cell assemblies to networks. This can potentially advance our understanding of complex brain functions and cognitive processes in health and disease. Commitment to open science and insights into large-scale computational neural dynamics could enhance brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Furthermore, understanding the underlying mechanisms of impaired large-scale neural computations and their interconnected microcircuit dynamics could lead to the identification of specific biomarkers, paving the way for more accurate diagnostic tools and targeted therapies for neurological disorders.

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Voltage-gated sodium channel (Na_V) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available Na_V-targeting antiseizure medications nonselectively inhibit the brain channels Na_V1.1, Na_V1.2, and Na_V1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule Na_V-targeting compounds. These compounds specifically target inhibition of the Na_V1.6 and Na_V1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against Na_V1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing Na_V1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg²⁺- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of Na_V1.2 and Na_V1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.

The delicate “Excitatory/Inhibitory balance” between neurons holds significance in neurodegenerative and neurodevelopmental diseases. With the ultimate goal of creating a faithful in vitro model of the human brain, in this study, we investigated the critical factor of heterogeneity, focusing on the interplay between excitatory glutamatergic (E) and inhibitory GABAergic (I) neurons in neural networks. We used high-density Micro-Electrode Arrays (MEA) with 2304 recording electrodes to investigate two neuronal culture configurations: 100% glutamatergic (100E) and 75% glutamatergic / 25% GABAergic (75E25I) neurons. This allowed us to comprehensively characterize the spontaneous electrophysiological activity exhibited by mature cultures at 56 Days in vitro, a time point in which the GABA shift has already occurred. We explored the impact of heterogeneity also through electrical stimulation, revealing that the 100E configuration responded reliably, while the 75E25I required more parameter tuning for improved responses. Chemical stimulation with BIC showed an increase in terms of firing and bursting activity only in the 75E25I condition, while APV and CNQX induced significant alterations on both dynamics and functional connectivity. Our findings advance understanding of diverse neuron interactions and their role in network activity, offering insights for potential therapeutic interventions in neurological conditions. Overall, this work contributes to the development of a valuable human-based in vitro system for studying physiological and pathological conditions, emphasizing the pivotal role of neuron diversity in neural network dynamics.

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months to years for reaching adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. Therefore, we designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons which identified multiple compounds that drive neuronal maturation including inhibitors of lysine-specific demethylase 1 and disruptor of telomerase-like 1 and activators of calcium-dependent transcription. A cocktail of four factors, GSK2879552, EPZ-5676, N-methyl-d-aspartate and Bay K 8644, collectively termed GENtoniK, triggered maturation across all parameters tested, including synaptic density, electrophysiology and transcriptomics. Maturation effects were further validated in cortical organoids, spinal motoneurons and non-neural lineages including melanocytes and pancreatic β-cells. The effects on maturation observed across a broad range of hPSC-derived cell types indicate that some of the mechanisms controlling the timing of human maturation might be shared across lineages.

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Highlights

• Lack of Trem2 in mice impairs hippocampal neuronal bioenergetics during development
• CA1 but not CA3 neurons show reduced mitochondrial mass and metabolism
• CA1 metabolic dysfunction is later accompanied by synaptic and network alterations
• A partial reduction in Trem2 is sufficient to alter neuronal metabolic fitness

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Summary

Triggering receptor expressed on myeloid cells 2 (Trem2) is a myeloid cell-specific gene expressed in brain microglia, with variants that are associated with neurodegenerative diseases, including Alzheimer’s disease. Trem2 is essential for microglia-mediated synaptic refinement, but whether Trem2 contributes to shaping neuronal development remains unclear. Here, we demonstrate that Trem2 plays a key role in controlling the bioenergetic profile of pyramidal neurons during development. In the absence of Trem2, developing neurons in the hippocampal cornus ammonis (CA)1 but not in CA3 subfield displayed compromised energetic metabolism, accompanied by reduced mitochondrial mass and abnormal organelle ultrastructure. This was paralleled by the transcriptional rearrangement of hippocampal pyramidal neurons at birth, with a pervasive alteration of metabolic, oxidative phosphorylation, and mitochondrial gene signatures, accompanied by a delay in the maturation of CA1 neurons. Our results unveil a role of Trem2 in controlling neuronal development by regulating the metabolic fitness of neurons in a region-specific manner.

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Highlights

• Mesoderm induction and patterning signals specify aSHF, pSHF, and FHF progenitors
• Progenitors sort, co-develop, and functionally connect in multi-chamber cardioids
• Multi-chamber cardioids coordinate contraction propagation and share a lumen
• Multi-chamber platform dissects genetic, teratogenic, and physiological defects

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Summary

The number one cause of human fetal death are defects in heart development. Because the human embryonic heart is inaccessible and the impacts of mutations, drugs, and environmental factors on the specialized functions of different heart compartments are not captured by in vitro models, determining the underlying causes is difficult. Here, we established a human cardioid platform that recapitulates the development of all major embryonic heart compartments, including right and left ventricles, atria, outflow tract, and atrioventricular canal. By leveraging 2D and 3D differentiation, we efficiently generated progenitor subsets with distinct first, anterior, and posterior second heart field identities. This advance enabled the reproducible generation of cardioids with compartment-specific in vivo-like gene expression profiles, morphologies, and functions. We used this platform to unravel the ontogeny of signal and contraction propagation between interacting heart chambers and dissect how mutations, teratogens, and drugs cause compartment-specific defects in the developing human heart.

Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2–3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.

Degeneration of photoreceptors in age-related macular degeneration (AMD) is associated with oxidative stress due to the intense aerobic metabolism of rods and cones that if not properly counterbalanced by endogenous antioxidant mechanisms can precipitate photoreceptor degeneration. In spite of being a priority eye disease for its high incidence in the elderly, no effective treatments for AMD exist. While systemic administration of antioxidants has been unsuccessful in slowing down degeneration, locally administered rare-earth nanoparticles were shown to be effective in preventing retinal photo-oxidative damage. However, because of inherent problems of dispersion in biological media, limited antioxidant power, and short lifetimes, these NPs are still confined to the preclinical stage. Here we propose platinum nanoparticles (PtNPs), potent antioxidant nanozymes, as a therapeutic tool for AMD. PtNPs exhibit high catalytic activity at minimal concentrations and protect primary neurons against oxidative insults and the ensuing apoptosis. We tested the efficacy of intravitreally injected PtNPs in preventing or mitigating light damage produced in dark-reared albino Sprague–Dawley rats by in vivo electroretinography (ERG) and ex vivo retina morphology and electrophysiology. We found that both preventive and postlesional treatments with PtNPs increased the amplitude of ERG responses to light stimuli. Ex vivo recordings demonstrated the selective preservation of ON retinal ganglion cell responses to light stimulation in lesioned retinas treated with PtNPs. PtNPs administered after light damage significantly preserved the number of photoreceptors and inhibited the inflammatory response to degeneration, while the preventive treatment had a milder effect. The data indicate that PtNPs can effectively break the vicious cycle linking oxidative stress, degeneration, and inflammation by exerting antioxidant and anti-inflammatory actions. The increased photoreceptor survival and visual performances in degenerated retinas, together with their high biocompatibility, make PtNPs a potential strategy to cure AMD.

The process of learning and memory formation in the hippocampus is a complex phenomenon involving changes in synaptic efficiency that shape the connections between associated neurons. Long-term potentiation (LTP) is a widely used approach for studying activity-dependent synaptic plasticity in the hippocampus. However, conventional methods of measuring LTP using brain slices and low-density microelectrode arrays (MEAs) have limitations in spatial mapping and induction of inter-experiential variability. To overcome these challenges, we implemented a large-scale ex-vivo electrophysiological platform using a high-density CMOS-based biosensor. This approach allows simultaneous recordings of network-wide evoked synaptic responses and LTP in the hippocampal circuit. It provides a platform for large-scale spatial mapping of hippocampal synaptic activity-dependent changes modulated by external electrical stimulation and resultant network LTP in the corresponding SC pathway. This approach can potentially instantiate a large-scale model for learning and memory by identifying network LTP-induced signaling cascades and their mechanisms with applications for aging, disease, and pharmacology in the context of synaptic plasticity.

Experiential richness creates tissue-level changes and synaptic plasticity as patterns emerge from rhythmic spatiotemporal activity of large interconnected neuronal assemblies. Despite numerous experimental and computational approaches at different scales, the precise impact of experience on network-wide computational dynamics remains inaccessible due to the lack of applicable large-scale recording methodology. We here demonstrate a large-scale multi-site biohybrid brain circuity on-CMOS-based biosensor with an unprecedented spatiotemporal resolution of 4096 microelectrodes, which allows simultaneous electrophysiological assessment across the entire hippocampal-cortical subnetworks from mice living in an enriched environment (ENR) and standard-housed (SD) conditions. Our platform, empowered with various computational analyses, reveals environmental enrichment's impacts on local and global spatiotemporal neural dynamics, firing synchrony, topological network complexity, and large-scale connectome. Our results delineate the distinct role of prior experience in enhancing multiplexed dimensional coding formed by neuronal ensembles and error tolerance and resilience to random failures compared to standard conditions. The scope and depth of these effects highlight the critical role of high-density, large-scale biosensors to provide a new understanding of the computational dynamics and information processing in multimodal physiological and experience-dependent plasticity conditions and their role in higher brain functions. Knowledge of these large-scale dynamics can inspire the development of biologically plausible computational models and computational artificial intelligence networks and expand the reach of neuromorphic brain-inspired computing into new applications.

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Exosomes, key mediators of intercellular transmission of pathogenic proteins, such as amyloid-beta and tau, significantly influence the progression and exacerbation of Alzheimer’s disease (AD) pathology. Present in a variety of biological fluids, including cerebrospinal fluid, blood, saliva, and nasal lavage fluid (NLF), exosomes underscore their potential as integral mediators of AD pathology. By serving as vehicles for disease-specific molecules, exosomes could unveil valuable insights into disease identification and progression. This study emphasizes the imperative to investigate the impacts of exosomes on neural networks to enhance our comprehension of intracerebral neuronal communication and its implications for neurological disorders like AD. After harvesting exosomes derived from NLF of 5XFAD mice, we utilized a high-density multielectrode array (HD-MEA) system, the novel technology enabling concurrent recordings from thousands of neurons in primary cortical neuron cultures and organotypic hippocampal slices. The ensuing results revealed a surge in neuronal firing rates and disoriented neural connectivity, reflecting the effects provoked by pathological amyloid-beta oligomer treatment. The local field potentials in the exosome-treated hippocampal brain slices also exhibited aberrant rhythmicity, along with an elevated level of current source density. While this research is an initial exploration, it highlights the potential of exosomes in modulating neural networks under AD conditions and endorses the HD-MEA as an efficacious tool for exosome studies.

In vertebrate vision, early retinal circuits divide incoming visual information into functionally opposite elementary signals: On and Off, transient and sustained, chromatic and achromatic. Together these signals can yield an efficient representation of the scene for transmission to the brain via the optic nerve. However, this long-standing interpretation of retinal function is based on mammals, and it is unclear whether this functional arrangement is common to all vertebrates. Here we show that male poultry chicks use a fundamentally different strategy to communicate information from the eye to the brain. Rather than using functionally opposite pairs of retinal output channels, chicks encode the polarity, timing, and spectral composition of visual stimuli in a highly correlated manner: fast achromatic information is encoded by Off-circuits, and slow chromatic information overwhelmingly by On-circuits. Moreover, most retinal output channels combine On- and Off-circuits to simultaneously encode, or multiplex, both achromatic and chromatic information. Our results from birds conform to evidence from fish, amphibians, and reptiles which retain the full ancestral complement of four spectral types of cone photoreceptors.

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Highlights

• Human retinal organoids express majority of miRNAs typically found in developing retina
• MiRNAs are differentially expressed in response to light
• Light-regulated miRNAs have a rapid turnover in human retinal tissue
• MiRNAs respond differently to distinct wavelengths of light

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Summary

Cells in the human retina must rapidly adapt to constantly changing visual stimuli. This fast adaptation to varying levels and wavelengths of light helps to regulate circadian rhythms and allows for adaptation to high levels of illumination, thereby enabling the rest of the visual system to remain responsive. It has been shown that retinal microRNA (miRNA) molecules play a key role in regulating these processes. However, despite extensive research using various model organisms, light-regulated miRNAs in human retinal cells remain unknown. Here, we aim to characterize these miRNAs. We generated light-responsive human retinal organoids that express miRNA families and clusters typically found in the retina. Using an in-house developed photostimulation device, we identified a subset of light-regulated miRNAs. Importantly, we found that these miRNAs are differentially regulated by distinct wavelengths of light and have a rapid turnover, highlighting the dynamic and adaptive nature of the human retina.

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The cerebellum is one of the most connected structures of the central nervous system and receives inputs over an extended frequency range. Nevertheless, the frequency dependence of cerebellar cortical processing remains elusive. In this work, we characterized cerebellar cortex responsiveness to mossy fibers activation at different frequencies and reconstructed the spread of activity in the sagittal and coronal planes of acute mouse cerebellar slices using a high-throughput high-density multielectrode array (HD-MEA). The enhanced spatiotemporal resolution of HD-MEA revealed the frequency dependence and spatial anisotropy of cerebellar activation. Mossy fiber inputs reached the Purkinje cell layer even at the lowest frequencies, but the efficiency of transmission increased at higher frequencies. These properties, which are likely to descend from the topographic organization of local inhibition, intrinsic electroresponsiveness, and short-term synaptic plasticity, are critical elements that have to be taken into consideration to define the computational properties of the cerebellar cortex and its pathological alterations.

(1) Background: The contribution of gene-specific variants for congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 on human cardiomyocyte (CM) differentiation and proliferation. (2) Methods: Taking advantage of our DAND5 patient-derived iPSC line, we used CRISPR-Cas9 gene-editing to generate a set of isogenic hiPSCs (DAND5-corrected and DAND5 full-mutant). The hiPSCs were differentiated into CMs, and RT-qPCR and immunofluorescence profiled the expression of cardiac markers. Cardiomyocyte proliferation was analysed by flow cytometry. Furthermore, we used a multi-electrode array (MEA) to study the functional electrophysiology of DAND5 hiPSC-CMs. (3) Results: The results indicated that hiPSC-CM proliferation is affected by DAND5 levels. Cardiomyocytes derived from a DAND5 full-mutant hiPSC line are more proliferative when compared with gene-corrected hiPSC-CMs. Moreover, parallel cardiac differentiations showed a differential cardiac gene expression profile, with upregulated cardiac progenitor markers in DAND5-KO hiPSC-CMs. Microelectrode array (MEA) measurements demonstrated that DAND5-KO hiPSC-CMs showed prolonged field potential duration and increased spontaneous beating rates. In addition, conduction velocity is reduced in the monolayers of hiPSC-CMs with full-mutant genotype. (4) Conclusions: The absence of DAND5 sustains the proliferation of hiPSC-CMs, which alters their electrophysiological maturation properties. These results using DAND5 hiPSC-CMs consolidate the findings of the in vitro and in vivo mouse models, now in a translational perspective. Altogether, the data will help elucidate the molecular mechanism underlying this human heart disease and potentiates new therapies for treating adult CHD.

Specific medications to combat cerebellar ataxias, a group of debilitating movement disorders characterized by difficulty with walking, balance and coordination, are still lacking. Notably, cerebellar microglial activation appears to be a common feature in different types of ataxic patients and rodent models. However, direct evidence that cerebellar microglial activation in vivo is sufficient to induce ataxia is still lacking. Here, by employing chemogenetic approaches to manipulate cerebellar microglia selectively and directly, we found that specific chemogenetic activation of microglia in the cerebellar vermis directly leads to ataxia symptoms in wild-type mice and aggravated ataxic motor deficits in 3-acetylpyridine (3-AP) mice, a classic mouse model of cerebellar ataxia. Mechanistically, cerebellar microglial proinflammatory activation induced by either chemogenetic M3D(Gq) stimulation or 3-AP modeling hyperexcites Purkinje cells (PCs), which consequently triggers ataxia. Blockade of microglia-derived TNF-α, one of the most important proinflammatory cytokines, attenuates the hyperactivity of PCs driven by microglia. Moreover, chemogenetic inhibition of cerebellar microglial activation or suppression of cerebellar microglial activation by PLX3397 and minocycline reduces the production of proinflammatory cytokines, including TNF-α, to effectively restore the overactivation of PCs and alleviate motor deficits in 3-AP mice. These results suggest that cerebellar microglial activation may aggravate the neuroinflammatory response and subsequently induce dysfunction of PCs, which in turn triggers ataxic motor deficits. Our findings thus reveal a causal relationship between proinflammatory activation of cerebellar microglia and ataxic motor symptoms, which may offer novel evidence for therapeutic intervention for cerebellar ataxias by targeting microglia and microglia-derived inflammatory mediators.

With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing.

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Alzheimer’s disease (AD) is a multifactorial disorder that affects cognitive functioning, behavior, and neuronal properties. The neuronal dysfunction is primarily responsible for cognitive decline in AD patients, with many causal factors including plaque accumulation of Aβ42. Neural hyperactivity induced by Aβ42 deposition causes abnormalities in neural networks, leading to alterations in synaptic activity and interneuron dysfunction. Even though neuroimaging techniques elucidated the underlying mechanism of neural connectivity, precise understanding at the cellular level is still elusive. Previous multielectrode array studies have examined the neuronal network modulation in in vitro cultures revealing the relevance of ion channels and the chemical modulators in the presence of Aβ42. In this study, we investigated neuronal connectivity and dynamic changes using a high-density multielectrode array, particularly looking at network-wide parameter changes over time. By comparing the neuronal network between normal and Aβ42treated neuronal cultures, it was possible to discover the direct pathological effect of the Aβ42 oligomer altering the network characteristics. The detrimental effects of the Aβ42 oligomer included not only a decline in spike activation but also a qualitative impairment in neural connectivity as well as a disorientation of dispersibility. As a result, this will improve our understanding of how neural networks are modified during AD progression.

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Mutations in PCDH19 gene, which encodes protocadherin-19 (PCDH19), cause Developmental and Epileptic Encephalopathy 9 (DEE9). Heterogeneous loss of PCDH19 expression in neurons is considered a key determinant of the disorder; however, how PCDH19 mosaic expression affects neuronal network activity and circuits is largely unclear. Here, we show that the hippocampus of Pcdh19 mosaic mice is characterized by structural and functional synaptic defects and by the presence of PCDH19-negative hyperexcitable neurons. Furthermore, global reduction of network firing rate and increased neuronal synchronization have been observed in different limbic system areas. Finally, network activity analysis in freely behaving mice revealed a decrease in excitatory/inhibitory ratio and functional hyperconnectivity within the limbic system of Pcdh19 mosaic mice. Altogether, these results indicate that altered PCDH19 expression profoundly affects circuit wiring and functioning, and provide new key to interpret DEE9 pathogenesis.

Background

In the cerebral cortex, disinhibited activity is characterized by propagating waves that spread across neural tissue. In this pathological state, a widely reported form of activity are spiral waves that travel in a circular pattern around a fixed spatial locus termed the center of mass. Spiral waves exhibit stereotypical activity and involve broad patterns of co-fluctuations, suggesting that they may be of lower complexity than healthy activity.

Results

To evaluate this hypothesis, we performed dense multi-electrode recordings of cortical networks where disinhibition was induced by perfusing a pro-epileptiform solution containing 4-Aminopyridine as well as increased potassium and decreased magnesium. Spiral waves were identified based on a spatially delimited center of mass and a broad distribution of instantaneous phases across electrodes. Individual waves were decomposed into “snapshots” that captured instantaneous neural activation across the entire network. The complexity of these snapshots was examined using a measure termed the participation ratio. Contrary to our expectations, an eigenspectrum analysis of these snapshots revealed a broad distribution of eigenvalues and an increase in complexity compared to baseline networks. A deep generative adversarial network was trained to generate novel exemplars of snapshots that closely captured cortical spiral waves. These synthetic waves replicated key features of experimental data including a tight center of mass, a broad eigenvalue distribution, spatially-dependent correlations, and a high complexity. By adjusting the input to the model, new samples were generated that deviated in systematic ways from the experimental data, thus allowing the exploration of a broad range of states from healthy to pathologically disinhibited neural networks.

Conclusions

Together, results show that the complexity of population activity serves as a marker along a continuum from healthy to disinhibited brain states. The proposed generative adversarial network opens avenues for replicating the dynamics of cortical seizures and accelerating the design of optimal neurostimulation aimed at suppressing pathological brain activity.

Introduction

FIRES is a rare epileptic encephalopathy induced by acute unremitting seizures that occur suddenly in healthy children or young adults after a febrile illness in the preceding 2 weeks. This condition results in high mortality, neurological disability, and drug-resistant epilepsy. The development of new therapeutics is hampered by the lack of validated experimental models. Our goal was to address this unmet need by providing a simple tool for rapid throughput screening of new therapies that target pathological inflammatory mechanisms in FIRES. The model was not intended to mimic the etiopathogenesis of FIRES which is still unknown, but to reproduce salient features of its clinical presentation such as the age, the cytokine storm and the refractoriness of epileptic activity to antiseizure medications (ASMs).

Methods

‍We refined an in vitro model of mouse hippocampal/temporal cortex acute slices where drug-resistant epileptic activity is induced by zero Mg²⁺/100 μM 4-aminopirydine. Clinical evidence suggests that acute unremitting seizures in FIRES are promoted by neuroinflammation triggered in the brain by the preceding infection. We mimicked this inflammatory component by exposing slices for 30 min to 10 μg/ml lipopolysaccharide (LPS).

Results

‍LPS induced a sustained neuroinflammatory response, as shown by increased mRNA levels of IL-1β, CXCL1 (IL-8), TNF, and increased IL-1β/IL-1Ra ratio. Epileptiform activity was exacerbated by neuroinflammation, also displaying increased resistance to maximal therapeutic concentrations of midazolam (100 μM), phenytoin (50 μM), sodium valproate (800 μM), and phenobarbital (100 μM). Treatment of LPS-exposed slices with two immunomodulatory drugs, a mouse anti-IL-6 receptor antibody (100 μM) corresponding to tocilizumab in humans, or anakinra (1.3 μM) which blocks the IL-1 receptor type 1, delayed the onset of epileptiform events and strongly reduced the ASM-resistant epileptiform activity evoked by neuroinflammation. These drugs were shown to reduce ASM-refractory seizures in FIRES patients.

Discussion

‍The neuroinflammatory component and the pharmacological responsiveness of epileptiform events provide a proof-of-concept validation of this in vitro model for the rapid selection of new treatments for acute ASM-refractory seizures in FIRES.

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Reactive astrocytes play an important role in neurological diseases, but their molecular and functional phenotypes in epilepsy are unclear. Here, we show that in patients with temporal lobe epilepsy (TLE) and mouse models of epilepsy, excessive lipid accumulation in astrocytes leads to the formation of lipid-accumulated reactive astrocytes (LARAs), a new reactive astrocyte subtype characterized by elevated APOE expression. Genetic knockout of APOE inhibited LARA formation and seizure activities in epileptic mice. Single-nucleus RNA sequencing in TLE patients confirmed the existence of a LARA subpopulation with a distinct molecular signature. Functional studies in epilepsy mouse models and human brain slices showed that LARAs promote neuronal hyperactivity and disease progression. Targeting LARAs by intervention with lipid transport and metabolism could thus provide new therapeutic options for drug-resistant TLE.

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Highlights

• GDF11 enables transition from trunk to sacral neural crest in human PSCs
• Posterior neural crest emerges via a neuro-mesodermal progenitor in vitro
• Vagal and sacral neural crest exhibit distinct migratory behaviors
• Combined vagal/sacral neural crest injection induces rescue in severe HSCR model

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Summary

The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors in vitro and in vivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung’s disease.

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Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.

Dystonia is a neurological syndrome that alters muscle control for voluntary movement and sustained posture. Although the basal ganglia play a role in dystonia, an abnormal cerebellar function is also involved. Deep brain stimulation (DBS) is a standard treatment option for drug-refractory dystonia, and the most promising targets are the Globus Pallidus internus (GPi) or the subthalamic nucleus. The mechanisms of DBS, however, are as yet unclear. In this context, we were interested in the impact of DBS on cerebellar activity and, specifically, the role of glutamatergic transmission in DBS-induced changes.

We explored this question in a genetic animal model of primary paroxysmal dystonia (dtsz mutant hamster) and appropriate controls, bilaterally implanted with bipolar DBS electrodes in the entopeduncular nucleus (homolog to the GPi in humans).

The dtsz hamster is known for alteration in the ganglia–thalamocortical circuit, cortico-striatal circuit, and limbic structures. These further support us in investigating the cerebellum network, especially the synapse plasticity and the expression of NR2A subunits of NMDA since we already know that the NR2A/NR2B ratio is increased in the striatum of dystonic hamsters.

To gauge cerebellar activity, parasagittal slices were recorded with a high-density microelectrode array (200 μm thick) (HD-MEA; 3Brain AG). To analyze the involvement of the glutamatergic system, cerebellar slices were treated with 50 μM of PEAQX, an antagonist selective GluN2A, and their activity compared to baseline recordings in Krebs solution (10 minutes, 2 mL/min, at room temperature).

Our previous results indicate that blocking the NMDA receptor with PEAQX might modulate the Purkinje cell spike firing concerning amplitude and frequency differentially between the DBS and sham-DBS groups.

Acknowledgment This study was supported by the German Research Foundation (DFG) within the Collaborative Research Centre (SFB 1270/1 ELAINE 299150580). We also thank Tina Sellmann and Anna Einsle for all their support.