ß-amyloid's neurotoxic mechanisms as defined by in vitro microelectrode arrays: a review.

Alzheimer's disease is characterised by the aggregation of ß-amyloid, a pathological feature believed to drive the neuronal loss and cognitive decline commonly seen in the disease. Given the growing prevalence of this progressive neurodegenerative disease, understanding the exact mechanisms underlying this process has become a top priority. Microelectrode arrays are commonly used for chronic, non-invasive recording of both spontaneous and evoked neuronal activity from diverse in vitro disease models and to evaluate therapeutic or toxic compounds. To date, microelectrode arrays have been used to investigate ß-amyloids' toxic effects, ß-amyloids role in specific pathological features and to assess pharmacological approaches to treat Alzheimer's disease. The versatility of microelectrode arrays means these studies use a variety of methods and investigate different disease models and brain regions. This review provides an overview of these studies, highlighting their disparities and presenting the status of the current literature. Despite methodological differences, the current literature indicates that ß-amyloid has an inhibitory effect on synaptic plasticity and induces network connectivity disruptions. ß-amyloid's effect on spontaneous neuronal activity appears more complex. Overall, the literature corroborates the theory that ß-amyloid induces neurotoxicity, having a progressive deleterious effect on neuronal signalling and plasticity. These studies also confirm that microelectrode arrays are valuable tools for investigating ß-amyloid pathology from a functional perspective, helping to bridge the gap between cellular and network pathology and disease symptoms. The use of microelectrode arrays provides a functional insight into Alzheimer's disease pathology which will aid in the development of novel therapeutic interventions.

Defining Cardiomyocyte Repolarization Response to Pharmacotherapy in Long-QT Syndrome Type 3.

BACKGROUND: Long-QT syndrome is a primary cardiac ion channelopathy predisposing a patient to ventricular arrhythmia through delayed repolarization on the resting ECG. We aimed to establish a patient-specific, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes model of long-QT syndrome type 3 (LQT3) using clustered regularly interspaced palindromic repeats (CRISPR/Cas9), for disease modeling and drug challenge. METHODS AND RESULTS: HiPSCs were generated from a patient with LQT3 harboring an SCN5A pathogenic variant (c.1231G>A; p.Val411Met), and an unrelated healthy control. The same SCN5A pathogenic variant was engineered into the background healthy control hiPSCs via CRISPR/Cas9 gene editing to generate a second disease model of LQT3 for comparison with an isogenic control. All 3 hiPSC lines were differentiated into cardiomyocytes. Both the patient-derived LQT3 (SCN5A+/-) and genetically engineered LQT3 (SCN5A+/-) hiPSC-derived cardiomyocytes showed significantly prolonged cardiomyocyte repolarization compared with the healthy control. Mexiletine, a cardiac voltage-gated sodium channel (NaV1.5) blocker, shortened repolarization in both patient-derived LQT3 and genetically engineered LQT3 hiPSC-derived cardiomyocytes, but had no effect in the control. Notably, calcium channel blockers nifedipine and verapamil showed a dose-dependent shortening of repolarization, rescuing the phenotype. Additionally, therapeutic drugs known to prolong the corrected QT in humans (ondansetron, clarithromycin, and sotalol) demonstrated this effect in vitro, but the LQT3 clones were not more disproportionately affected compared with the control. CONCLUSIONS: We demonstrated that patient-derived and genetically engineered LQT3 hiPSC-derived cardiomyocytes faithfully recapitulate pathologic characteristics of LQT3. The clinical significance of such an in vitro model is in the exploration of novel therapeutic strategies, stratifying drug adverse reaction risk and potentially facilitating a more targeted, patient-specific approach in high-risk patients with LQT3.

Analyzing the transient response dynamics of long-term depression in the mouse auditory cortex in vitro through multielectrode-array-based spatiotemporal recordings.

In the auditory cortex, synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), plays crucial roles in information processing and adaptation to the auditory environment. Previous rodent studies have shown lifelong cortical map plasticity, even beyond the critical period of development. While thalamocortical synapses exhibit LTD during the critical period, little is known about LTD in the cortico-cortical connections of the adult mouse auditory cortex. Here, we investigated the transient response dynamics of LTD in layers 2-5 of the mouse auditory cortex following tetanic stimulation (TS) to layer 4. To characterize LTD properties, we developed a recording protocol to monitor activity levels at multiple sites, including those more than 0.45 mm from the TS site. This allowed us to distinguish LTD-induced reductions in neural excitability from other types, including neural activity depletion. Our findings revealed that LTD induced in layer 4 persisted for over 40-min post-TS, indicating robust cortico-cortical LTD. Using electrophysiological data and a modified synaptic model, we identified key receptors involved in synaptic plasticity and their effects on response dynamics, proposing a method for studying LTD in the mature mouse auditory cortex. Particularly, by employing a simple dynamical model, we analyzed and discussed the involvement of key receptors during the transient period of LTD. This study expands our understanding of synaptic plasticity in the mature mouse auditory cortex beyond the critical period, potentially informing future treatments for hearing disorders.

A high-density multi-electrode platform examining the effects of radiation on in vitro cortical networks.

Radiation therapy and stereotactic radiosurgery are common treatments for brain malignancies. However, the impact of radiation on underlying neuronal circuits is poorly understood. In the prefrontal cortex (PFC), neurons communicate via action potentials that control cognitive processes, thus it is important to understand the impact of radiation on these circuits. Here we present a novel protocol to investigate the effect of radiation on the activity and survival of PFC networks in vitro. Escalating doses of radiation were applied to PFC slices using a robotic radiosurgery platform at a standard dose rate of 10 Gy/min. High-density multielectrode array recordings of radiated slices were collected to capture extracellular activity across 4,096 channels. Radiated slices showed an increase in firing rate, functional connectivity, and complexity. Graph-theoretic measures of functional connectivity were altered following radiation. These results were compared to pharmacologically induced epileptic slices where neural complexity was markedly elevated, and functional connections were strong but remained spatially focused. Finally, propidium iodide staining revealed a dose-dependent effect of radiation on apoptosis. These findings provide a novel assay to investigate the impacts of clinically relevant doses of radiation on brain circuits and highlight the acute effects of escalating radiation doses on PFC neurons.

Homeostatic Regulation of Spike Rate within Bursts in Two Distinct Preparations.

Homeostatic plasticity represents a set of mechanisms thought to stabilize some function of neural activity. Here, we identified the specific features of cellular or network activity that were maintained after the perturbation of GABAergic blockade in two different systems: mouse cortical neuronal cultures where GABA is inhibitory and motoneurons in the isolated embryonic chick spinal cord where GABA is excitatory (males and females combined in both systems). We conducted a comprehensive analysis of various spiking activity characteristics following GABAergic blockade. We observed significant variability in many features after blocking GABAA receptors (e.g., burst frequency, burst duration, overall spike frequency in culture). These results are consistent with the idea that neuronal networks achieve activity goals using different strategies (degeneracy). On the other hand, some features were consistently altered after receptor blockade in the spinal cord preparation (e.g., overall spike frequency). Regardless, these features did not express strong homeostatic recoveries when tracking individual preparations over time. One feature showed a consistent change and homeostatic recovery following GABAA receptor block. We found that spike rate within a burst (SRWB) increased after receptor block in both the spinal cord preparation and cortical cultures and then returned to baseline within hours. These changes in SRWB occurred at both single cell and population levels. Our findings indicate that the network prioritizes the burst spike rate, which appears to be a variable under tight homeostatic regulation. The result is consistent with the idea that networks can maintain an appropriate behavioral response in the face of challenges.

Cortical Tagged Synaptic Long-Term Depression in the Anterior Cingulate Cortex of Adult Mice.

The anterior cingulate cortex (ACC) is a key cortical region for pain perception and emotion. Different forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), have been reported in the ACC. Synaptic tagging of LTP plays an important role in hippocampus-related associative memory. In this study, we demonstrate that synaptic tagging of LTD is detected in the ACC of adult male and female mice. This form of tagged LTD requires the activation of metabotropic glutamate receptor subtype 1 (mGluR1). The induction of tagged LTD is time-related with the strongest tagged LTD appearing when the interval between two independent stimuli is 30 min. Inhibitors of mGluR1 blocked the induction of tagged LTD; however, blocking N-methyl-d-aspartate receptors did not affect the induction of tagged LTD. Nimodipine, an inhibitor of L-type voltage-gated calcium channels, also blocked tagged LTD. In an animal model of amputation, we found that tagged LTD was either reduced or completely blocked. Together with our previous report of tagged LTP in the ACC, this study strongly suggests that excitatory synapses in the adult ACC are highly plastic. The biphasic tagging of synaptic transmission provides a new form of heterosynaptic plasticity in the ACC which has functional and pathophysiological significance in phantom pain.

Developmental effect of RASopathy mutations on neuronal network activity on a chip.

RASopathies are a group of genetic disorders caused by mutations in genes encoding components and regulators of the RAS/MAPK signaling pathway, resulting in overactivation of signaling. RASopathy patients exhibit distinctive facial features, cardiopathies, growth and skeletal abnormalities, and varying degrees of neurocognitive impairments including neurodevelopmental delay, intellectual disabilities, or attention deficits. At present, it is unclear how RASopathy mutations cause neurocognitive impairment and what their neuron-specific cellular and network phenotypes are. Here, we investigated the effect of RASopathy mutations on the establishment and functional maturation of neuronal networks. We isolated cortical neurons from RASopathy mouse models, cultured them on multielectrode arrays and performed longitudinal recordings of spontaneous activity in developing networks as well as recordings of evoked responses in mature neurons. To facilitate the analysis of large and complex data sets resulting from long-term multielectrode recordings, we developed MATLAB-based tools for data processing, analysis, and statistical evaluation. Longitudinal analysis of spontaneous network activity revealed a convergent developmental phenotype in neurons carrying the gain-of-function Noonan syndrome-related mutations Ptpn11 D61Y and Kras V14l. The phenotype was more pronounced at the earlier time points and faded out over time, suggesting the emergence of compensatory mechanisms during network maturation. Nevertheless, persistent differences in excitatory/inhibitory balance and network excitability were observed in mature networks. This study improves the understanding of the complex relationship between genetic mutations and clinical manifestations in RASopathies by adding insights into functional network processes as an additional piece of the puzzle.

Mesenchymal Stem Cells Promote an Increase in Neuronal Oscillation via Glutamate Tonic Release.

Mesenchymal stromal cells (MSCs) hold therapeutic potential for neurological disorders, but their impact on neuronal activity remains unclear. We investigated the effects of SB623 cells (Notch-1 intracellular domain-transfected MSCs) and parental MSCs on human induced pluripotent stem cell (iPSC)-derived neurons using multi-electrode arrays. SB623 cells significantly increased neuronal activity and oscillation in a dose-dependent manner, surpassing astrocytes in promoting network bursts. Strikingly, glutamatergic neurons showed a rapid increase in activity and bursts compared to GABAergic neurons, suggesting glutamate release from SB623 cells. We confirmed this by finding high glutamate levels in SB623 cell conditioned medium, which were reduced by glutaminase inhibition. Glutamate release was further implicated by the reduced excitability in co-cultures with astrocytes, known glutamate scavengers. Our findings reveal a novel mechanism for MSCs: promoting neuronal activity and network formation through tonic glutamate release, with potential implications for MSC-based therapies.

Vertebrates on a Chip: Noninvasive Electrical and Optical Mapping of Whole Brain Activity Associated with Pharmacological Treatments.

Mapping brain activities is necessary for understanding brain physiology and discovering new treatments for neurological disorders. Such efforts have greatly benefited from the advancement in technologies for analyzing neural activity with improving temporal or spatial resolution. Here, we constructed a multielectrode array based brain activity mapping (BAM) system capable of stabilizing and orienting zebrafish larvae for recording electroencephalogram (EEG) like local field potential (LFP) signals and brain-wide calcium dynamics in awake zebrafish. Particularly, we designed a zebrafish trap chip that integrates with an eight-by-eight surface electrode array, so that brain electrophysiology can be noninvasively recorded in an agarose-free and anesthetic-free format with a high temporal resolution of 40 µs, matching the capability typically achieved by invasive LFP recording. Benefiting from the specially designed hybrid system, we can also conduct calcium imaging directly on immobilized awake larval zebrafish, which further supplies us with high spatial resolution brain-wide activity data. All of these innovations reconcile the limitations of sole LFP recording or calcium imaging, emphasizing a synergy of combining electrical and optical modalities within one unified device for activity mapping across a whole vertebrate brain with both improved spatial and temporal resolutions. The compatibility with in vivo drug treatment further makes it suitable for pharmacology studies based on multimodal measurement of brain-wide physiology.

Enhanced electrophysiological activity and neurotoxicity screening of environmental chemicals using 3D neurons from human neural precursor cells purified with PSA-NCAM.

The assessment of neurotoxicity for environmental chemicals is of utmost importance in ensuring public health and environmental safety. Multielectrode array (MEA) technology has emerged as a powerful tool for assessing disturbances in the electrophysiological activity. Although human embryonic stem cell (hESC)-derived neurons have been used in MEA for neurotoxicity screening, obtaining a substantial and sufficiently active population of neurons from hESCs remains challenging. In this study, we successfully differentiated neurons from a large population of human neuronal precursor cells (hNPC) purified using a polysialylated neural cell adhesion molecule (PSA-NCAM), referred to as hNPCPSA-NCAM+. The functional characterization demonstrated that hNPCPSA-NCAM+-derived neurons improve functionality by enhancing electrophysiological activity compared to total hNPC-derived neurons. Furthermore, three-dimensional (3D) neurons derived from hNPCPSA-NCAM+ exhibited reduced maturation time and enhanced electrophysiological activity on MEA. We employed subdivided population analysis of active mean firing rate (MFR) based on electrophysiological intensity to characterize the electrophysiological properties of hNPCPSA-NCAM+-3D neurons. Based on electrophysiological activity including MFR and burst parameters, we evaluated the sensitivity of hNPCPSA-NCAM+-3D neurons on MEA to screen both inhibitory and excitatory neuroactive environmental chemicals. Intriguingly, electrophysiologically active hNPCPSA-NCAM+-3D neurons demonstrated good sensitivity to evaluate neuroactive chemicals, particularly in discriminating excitatory chemicals. Our findings highlight the effectiveness of MEA approaches using hNPCPSA-NCAM+-3D neurons in the assessment of neurotoxicity associated with environmental chemicals. Furthermore, we emphasize the importance of selecting appropriate signal intensity thresholds to enhance neurotoxicity prediction and screening of environmental chemicals.

Phase-amplitude coupling detection and analysis of human 2-dimensional neural cultures in multi-well microelectrode array in vitro.

BACKGROUND: Human induced pluripotent stem cell (hiPSC)- derived neurons offer the possibility of studying human-specific neuronal behaviors in physiologic and pathologic states in vitro. It is unclear whether cultured neurons can achieve the fundamental network behaviors required to process information in the brain. Investigating neuronal oscillations and their interactions, as occurs in cross-frequency coupling (CFC), addresses this question. NEW METHODS: We examined whether networks of two-dimensional (2D) cultured hiPSC-derived cortical neurons grown with hiPSC-derived astrocytes on microelectrode array plates recapitulate the CFC that is present in vivo. We employed the modulation index method for detecting phase-amplitude coupling (PAC) and used offline spike sorting to analyze the contribution of single neuron spiking to network behavior. RESULTS: We found that PAC is present, the degree of PAC is specific to network structure, and it is modulated by external stimulation with bicuculline administration. Modulation of PAC is not driven by single neurons, but by network-level interactions. COMPARISON WITH EXISTING METHODS: PAC has been demonstrated in multiple regions of the human cortex as well as in organoids. This is the first report of analysis demonstrating the presence of coupling in 2D cultures. CONCLUSION: CFC in the form of PAC analysis explores communication and integration between groups of neurons and dynamical changes across networks. In vitro PAC analysis has the potential to elucidate the underlying mechanisms as well as capture the effects of chemical, electrical, or ultrasound stimulation; providing insight into modulation of neural networks to treat nervous system disorders in vivo.

Phenotypic analysis of multielectrode array EEG biomarkers in developing and adult male Fmr1 KO mice.

Fragile X Syndrome (FXS) is a leading known genetic cause of intellectual disability with symptoms that include increased anxiety and social and sensory processing deficits. Recent electroencephalographic (EEG) studies in humans with FXS have identified neural oscillation deficits that include increased resting state gamma power, increased amplitude of auditory evoked potentials, and reduced phase locking of sound-evoked gamma oscillations. Similar EEG phenotypes are present in mouse models of FXS, but very little is known about the development of such abnormal responses. In the current study, we employed a 30-channel mouse multielectrode array (MEA) system to record and analyze resting and stimulus-evoked EEG signals in male P21 and P91 WT and Fmr1 KO mice. This led to several novel findings. First, P91, but not P21, Fmr1 KO mice have significantly increased resting EEG power in the low- and high-gamma frequency bands. Second, both P21 and P91 Fmr1 KO mice have markedly attenuated inter-trial phase coherence (ITPC) to spectrotemporally dynamic auditory stimuli as well as to 40 Hz and 80 Hz auditory steady-state response (ASSR) stimuli. This suggests abnormal temporal processing from early development that may lead to abnormal speech and language function in FXS. Third, we found hemispheric asymmetry of fast temporal processing in the mouse auditory cortex in WT but not Fmr1 KO mice. Together, these findings define a set of EEG phenotypes in young and adult mice that can serve as translational targets for genetic and pharmacological manipulation in phenotypic rescue studies.

XMEA: A New Hybrid Diamond Multielectrode Array for the In Situ Assessment of the Radiation Dose Enhancement by Nanoparticles.

This work presents a novel multielectrode array (MEA) to quantitatively assess the dose enhancement factor (DEF) produced in a medium by embedded nanoparticles. The MEA has 16 nanocrystalline diamond electrodes (in a cell-culture well), and a single-crystal diamond divided into four quadrants for X-ray dosimetry. DEF was assessed in water solutions with up to a 1000 µg/mL concentration of silver, platinum, and gold nanoparticles. The X-ray detectors showed a linear response to radiation dose (r2 ≥ 0.9999). Overall, platinum and gold nanoparticles produced a dose enhancement in the medium (maximum of 1.9 and 3.1, respectively), while silver nanoparticles produced a shielding effect (maximum of 37%), lowering the dose in the medium. This work shows that the novel MEA can be a useful tool in the quantitative assessment of radiation dose enhancement due to nanoparticles. Together with its suitability for cells' exocytosis studies, it proves to be a highly versatile device for several applications.

In Vitro Pharmacological Modulation of PIEZO1 Channels in Frontal Cortex Neuronal Networks.

PIEZO1 is a mechanosensitive ion channel expressed in various organs, including but not limited to the brain, heart, lungs, kidneys, bone, and skin. PIEZO1 has been implicated in astrocyte, microglia, capillary, and oligodendrocyte signaling in the mammalian cortex. Using murine embryonic frontal cortex tissue, we examined the protein expression and functionality of PIEZO1 channels in cultured networks leveraging substrate-integrated microelectrode arrays (MEAs) with additional quantitative results from calcium imaging and whole-cell patch-clamp electrophysiology. MEA data show that the PIEZO1 agonist Yoda1 transiently enhances the mean firing rate (MFR) of single units, while the PIEZO1 antagonist GsMTx4 inhibits both spontaneous activity and Yoda1-induced increase in MFR in cortical networks. Furthermore, calcium imaging experiments revealed that Yoda1 significantly increased the frequency of calcium transients in cortical cells. Additionally, in voltage clamp experiments, Yoda1 exposure shifted the cellular reversal potential towards depolarized potentials consistent with the behavior of PIEZO1 as a non-specific cation-permeable channel. Our work demonstrates that murine frontal cortical neurons express functional PIEZO1 channels and quantifies the electrophysiological effects of channel activation in vitro. By quantifying the electrophysiological effects of PIEZO1 activation in vitro, our study establishes a foundation for future investigations into the role of PIEZO1 in neurological processes and potential therapeutic applications targeting mechanosensitive channels in various physiological contexts.

Calcium-Dependent Hyperexcitability in Human Stem Cell-Derived Rett Syndrome Neuronal Networks.

Background: Mutations in MECP2 predominantly cause Rett syndrome and can be modeled in vitro using human stem cell-derived neurons. Patients with Rett syndrome have signs of cortical hyperexcitability, such as seizures. Human stem cell-derived MECP2 null excitatory neurons have smaller soma size and reduced synaptic connectivity but are also hyperexcitable due to higher input resistance. Paradoxically, networks of MECP2 null neurons show a decrease in the frequency of network bursts consistent with a hypoconnectivity phenotype. Here, we examine this issue. Methods: We reanalyzed multielectrode array data from 3 isogenic MECP2 cell line pairs recorded over 6 weeks (n = 144). We used a custom burst detection algorithm to analyze network events and isolated a phenomenon that we termed reverberating super bursts (RSBs). To probe potential mechanisms of RSBs, we conducted pharmacological manipulations using bicuculline, EGTA-AM, and DMSO on 1 cell line (n = 34). Results: RSBs, often misidentified as single long-duration bursts, consisted of a large-amplitude initial burst followed by several high-frequency, low-amplitude minibursts. Our analysis revealed that MECP2 null networks exhibited increased frequency of RSBs, which produced increased bursts compared with isogenic controls. Bicuculline or DMSO treatment did not affect RSBs. EGTA-AM selectively eliminated RSBs and rescued network burst dynamics. Conclusions: During early development, MECP2 null neurons are hyperexcitable and produce hyperexcitable networks. This may predispose them to the emergence of hypersynchronic states that potentially translate into seizures. Network hyperexcitability depends on asynchronous neurotransmitter release that is likely driven by presynaptic Ca2+ and can be rescued by EGTA-AM to restore typical network dynamics.

Increased Dentate Gyrus Excitability in the Intrahippocampal Kainic Acid Mouse Model for Temporal Lobe Epilepsy.

The intrahippocampal kainic acid (IHKA) mouse model is an extensively used in vivo model to investigate the pathophysiology of mesial temporal lobe epilepsy (mTLE) and to develop novel therapies for drug-resistant epilepsy. It is characterized by profound hippocampal sclerosis and spontaneously occurring seizures with a major role for the injected damaged hippocampus, but little is known about the excitability of specific subregions. The purpose of this study was to electrophysiologically characterize the excitability of hippocampal subregions in the chronic phase of the induced epilepsy in the IHKA mouse model. We recorded field postsynaptic potentials (fPSPs) after electrical stimulation in the CA1 region and in the dentate gyrus (DG) of hippocampal slices of IHKA and healthy mice using a multielectrode array (MEA). In the DG, a significantly steeper fPSP slope was found, reflecting higher synaptic strength. Population spikes were more prevalent with a larger spatial distribution in the IHKA group, reflecting a higher degree of granule cell output. Only minor differences were found in the CA1 region. These results point to increased neuronal excitability in the DG but not in the CA1 region of the hippocampus of IHKA mice. This method, in which the excitability of hippocampal slices from IHKA mice is investigated using a MEA, can now be further explored as a potential new model to screen for new interventions that can restore DG function and potentially lead to novel therapies for mTLE.

Human cytomegalovirus induces significant structural and functional changes in terminally differentiated human cortical neurons.

IMPORTANCE: Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that can cause serious neurological deficits in infants experiencing an in utero infection. Also, as a life-long infection, HCMV has been associated with several diseases in the adult brain. HCMV is known to infect early neural progenitor cells, but whether it also infects terminally differentiated neurons is still debated. Here, we differentiated human-induced pluripotent stem cells into neurons for 84-120 days to test the ability of HCMV to infect terminally differentiated neurons and assess the downstream functional consequences. We discovered that mature human neurons are highly permissive to HCMV infection, exhibited late replication hallmarks, and produced infectious virus. Moreover, infection in terminally differentiated neurons essentially eliminated neuron function. These results demonstrate that terminally differentiated human neurons are permissive to HCMV infection, which can significantly alter both structural and functional features of this mature neuron population.

Islets-on-Chip: A Tool for Real-Time Assessment of Islet Function Prior to Transplantation.

Islet transplantation improves metabolic control in patients with unstable type 1 diabetes. Clinical outcomes have been improving over the last decade, and the widely used beta-score allows the evaluation of transplantation results. However, predictive pre-transplantation criteria of islet quality for clinical outcomes are lacking. In this proof-of-concept study, we examined whether characterization of the electrical activity of donor islets could provide a criterion. Aliquots of 8 human donor islets from the STABILOT study, sampled from islet preparations before transplantation, were characterized for purity and split for glucose-induced insulin secretion and electrical activity using multi-electrode-arrays. The latter tests glucose concentration dependencies, biphasic activity, hormones, and drug effects (adrenalin, GLP-1, glibenclamide) and provides a ranking of CHIP-scores from 1 to 6 (best) based on electrical islet activity. The analysis was performed online in real time using a dedicated board or offline. Grouping of beta-scores and CHIP-scores with high, intermediate, and low values was observed. Further analysis indicated correlation between CHIP-score and beta-score, although significance was not attained (R = 0.51, p = 0.1). This novel approach is easily implantable in islet isolation units and might provide means for the prediction of clinical outcomes. We acknowledge the small cohort size as the limitation of this pilot study.

Electrophysiological characterisation of iPSC-derived human ß-like cells and an SLC30A8 disease model.

iPSC-derived human ß-like cells (BLC) hold promise for both therapy and disease modelling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single cell electrophysiological tools to evaluate BLCs functions. The Multi-Electrode Arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs are electrically coupled, produce slow potential (SP) signals like primary ß-cells that are closely linked to insulin secretion. We also used high-resolution single-cell patch-clamp measurements to capture the exocytotic properties, and characterize voltage-gated sodium and calcium currents. These were comparable to those in primary ß and EndoC-ßH1 cells. The KATP channel conductance is greater than in human primary ß cells which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes protective SLC30A8 allele (p.Lys34Serfs*50) and found that BLCs with this allele have stronger electrical coupling. Our data suggest that with an adapted approach BLCs from pioneer protocol can be used to evaluate the functional impact of genetic variants on ß-cell function and coupling.

Transcriptional Dysregulation and Impaired Neuronal Activity in FMR1 Knock-Out and Fragile X Patients' iPSC-Derived Models.

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.