Inferring monosynaptic connections from paired dendritic spine Ca2+ imaging and large-scale recording of extracellular spiking

Objective: Techniques to identify monosynaptic connections between neurons have been vital for neuroscience research, facilitating important advancements concerning network topology, synaptic plasticity, and synaptic integration, among others. Approach: Here, we introduce a novel approach to identify and monitor monosynaptic connections using high-resolution dendritic spine Ca2+ imaging combined with simultaneous large-scale recording of extracellular electrical activity by means of high-density microelectrode arrays. Main results: We introduce an easily adoptable analysis pipeline that associates the imaged spine with its presynaptic unit and test it on in vitro recordings. The method is further validated and optimized by simulating synaptically-evoked spine Ca2+ transients based on measured spike trains in order to obtain simulated ground-truth connections. Significance: The proposed approach offers unique advantages as (a) it can be used to identify monosynaptic connections with an accurate localization of the synapse within the dendritic tree, (b) it provides precise information of presynaptic spiking, and (c) postsynaptic spine Ca2+ signals and, finally, (d) the non-invasive nature of the proposed method allows for long-term measurements. The analysis toolkit together with the rich data sets that were acquired are made publicly available for further exploration by the research community.ISSN:1741-2560ISSN:1741-255

Electrophysiological Phenotype Characterization of Human iPSC‐Derived Neuronal Cell Lines by Means of High‐Density Microelectrode Arrays

Recent advances in the field of cellular reprogramming have opened a route to studying the fundamental mechanisms underlying common neurological disorders. High‐density microelectrode‐arrays (HD‐MEAs) provide unprecedented means to study neuronal physiology at different scales, ranging from network through single‐neuron to subcellular features. In this work, HD‐MEAs are used in vitro to characterize and compare human induced‐pluripotent‐stem‐cell‐derived dopaminergic and motor neurons, including isogenic neuronal lines modeling Parkinson's disease and amyotrophic lateral sclerosis. Reproducible electrophysiological network, single‐cell and subcellular metrics are used for phenotype characterization and drug testing. Metrics, such as burst shape and axonal velocity, enable the distinction of healthy and diseased neurons. The HD‐MEA metrics can also be used to detect the effects of dosing the drug retigabine to human motor neurons. Finally, it is shown that the ability to detect drug effects and the observed culture‐to‐culture variability critically depend on the number of available recording electrodes. © 2021 Wiley-VCHISSN:2701-019

Mechanical stimulation and electrophysiological monitoring at subcellular resolution reveals differential mechanosensation of neurons within networks

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control.ISSN:1748-3387ISSN:1748-339

Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity in targeting phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or β2-glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by–among other factors–viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL with prothrombin (PT). The strength of the antibody response against SARS-CoV-2 was further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity, and aPL against PT in patients with SARS-CoV-2Author summary: An infection with SARS-CoV-2 affects patients differently. While some individuals do not develop any symptoms at all, others become seriously ill, displaying signs of hypercoagulation with involvement of different organ systems. The immune response triggered upon infection may comprise both protective as well as detrimental elements. Among the latter, antibodies targeting a wide range of self-proteins have been evidenced. Our research has focused on autoantibodies targeting phospholipids and phospholipid-binding proteins, held responsible for an increased tendency to thrombosis in an autoimmune disease called antiphospholipid syndrome. Using cohorts from different centres, including patients of mixed disease severities, we found that IgM antibodies targeting prothrombin and β2-glycoprotein I are enriched upon infection. Using a linear mixed-effects model, we further established that anti-prothrombin IgM autoantibodies emerge in proportion to the strength of the antibody response elicited against SARS-CoV-2 proteins, with disease severity and sex as additional contributors. Further research is warranted to investigate the relation between infection with SARS-CoV-2, hypercoagulation, and the presence of autoantibodies directed against prothrombin.ISSN:1553-7374ISSN:1553-736

Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity in targeting phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or β2-glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by–among other factors–viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL with prothrombin (PT). The strength of the antibody response against SARS-CoV-2 was further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity, and aPL against PT in patients with SARS-CoV-2