Julia Izsak, Henrik Seth, Margarita Iljin, Stephan Theiss, Hans Ågren, Keiko Funa, Ludwig Aigner, Eric Hanse &  Sebastian Illes 

Abstract

Lithium salts are used as mood-balancing medication prescribed to patients suffering from neuropsychiatric disorders, such as bipolar disorder and major depressive disorder. Lithium salts cross the blood-brain barrier and reach the brain parenchyma within few hours after oral application, however, how lithium influences directly human neuronal function is unknown. We applied patch–clamp and microelectrode array technology on human induced pluripotent stem cell (iPSC)-derived cortical neurons acutely exposed to therapeutic (<1 mM) and overdose concentrations (>1 mM) of lithium chloride (LiCl) to assess how therapeutically effective and overdose concentrations of LiCl directly influence human neuronal electrophysiological function at the synapse, single-cell, and neuronal network level. We describe that human iPSC-cortical neurons exposed to lithium showed an increased neuronal activity under all tested concentrations. Furthermore, we reveal a lithium-induced, concentration-dependent, transition of regular synchronous neuronal network activity using therapeutically effective concentration (<1 mM LiCl) to epileptiform-like neuronal discharges using overdose concentration (>1 mM LiCl). The overdose concentration lithium-induced epileptiform-like activity was similar to the epileptiform-like activity caused by the GABA_A-receptor antagonist. Patch–clamp recordings reveal that lithium reduces action potential threshold at all concentrations, however, only overdose concentration causes increased frequency of spontaneous AMPA-receptor mediated transmission. By applying the AMPA-receptor antagonist and anti-epileptic drug Perampanel, we demonstrate that Perampanel suppresses lithium-induced epileptiform-like activity in human cortical neurons. We provide insights in how therapeutically effective and overdose concentration of lithium directly influences human neuronal function at synapse, a single neuron, and neuronal network levels. Furthermore, we provide evidence that Perampanel suppresses pathological neuronal discharges caused by overdose concentrations of lithium in human neurons.

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Julia Izsak, Henrik Seth, Stephan Theiss, Eric Hanse, Sebastian Illes

Human induced pluripotent stem cell (hiPSC)-derived in vitro neural and organoid models resemble fetal, rather than adult brain properties, indicating that currently applied cultivation media and supplements are insufficient to achieve neural maturation beyond the fetal stage. In vivo, cerebrospinal fluid molecules are regulating the transition of the immature fetal human brain into a mature adult brain. By culturing hiPSC-3D neural aggregates in human cerebrospinal fluid (hCSF) obtained from healthy adult individuals, we demonstrate that hCSF rapidly triggers neurogenesis, gliogenesis, synapse formation, neurite outgrowth, suppresses proliferation of residing neural stem cells, and results in the formation of synchronously active neuronal circuits in vitro within 3 days. Thus, a physiologically relevant and adult brain-like milieu triggers maturation of hiPSC-3D neural aggregates into highly functional neuronal circuits in vitro. The approach presented here opens a new avenue to identify novel physiological factors for the improvement of hiPSC neural in vitro models.

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Julia Izsak, Henrik Seth, Mats Andersson, Dzeneta Vizlin-Hodzic, Stephan Theiss, Eric Hanse, Hans Ågren, Keiko Funa, Sebastian Illes

Reproducibly generating human induced pluripotent stem cell-based functional neuronal circuits, solely obtained from single individuals, poses particular challenges to achieve personalized and patient specific functional neuronal in vitro models. A hallmark of functional neuronal assemblies, synchronous neuronal activity, can be non-invasively studied by microelectrode array (MEA) technology, reliably capturing physiological and pathophysiological aspects of human brain function. In our here presented manuscript, we demonstrate a procedure to generate 3D neural aggregates comprising astrocytes, oligodendroglial cells, and neurons obtained from the same human tissue sample. Moreover, we demonstrate the robust ability of those neurons to create a highly synchronously active neuronal network within 3 weeks in vitro, without additionally applied astrocytes. The fusion of MEA-technology with functional neuronal circuits solely obtained from one individual’s cells represent isogenic person-specific human neuronal sensor chips that pave the way for specific personalized in vitro neuronal networks as well as neurological and neuropsychiatric disease modeling.

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Måns Henningson, Sebastian Illes

Abstract

Multi-electrode arrays (MEA) are increasingly used to investigate spontaneous neuronal network activity. The recorded signals comprise several distinct components: Apart from artifacts without biological significance, one can distinguish between spikes (action potentials) and subthreshold fluctuations (local fields potentials). Here we aim to develop a theoretical model that allows for a compact and robust characterization of subthreshold fluctuations in terms of a Gaussian statistical field theory in two spatial and one temporal dimension. What is usually referred to as the driving noise in the context of statistical physics is here interpreted as a representation of the neural activity. Spatial and temporal correlations of this activity give valuable information about the connectivity in the neural tissue. We apply our methods on a dataset obtained from MEA-measurements in an acute hippocampal brain slice from a rat. Our main finding is that the empirical correlation functions indeed obey the logarithmic behavior that is a general feature of theoretical models of this kind. We also find a clear correlation between the activity and the occurrence of spikes. Another important insight is the importance of correctly separating out certain artifacts from the data before proceeding with the analysis.

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Sebastian Illes, Martin Jakab, Felix Beyer, Renate Gelfert, Sébastien Couillard-Despres, Alfons Schnitzler, Markus Ritter, Ludwig Aigner

Summary

Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks.

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Frauke Otto MD, Sebastian Illes MSc, Jessica Opatz PhD, Maurice Laryea PhD, Stephan Theiss MSc, Hans-Peter Hartung MD, Alfons Schnitzler MD, Mario Siebler MD, Marcel Dihné MD

Neurological diseases frequently induce pathological changes of cerebrospinal fluid (CSF) that might secondarily influence brain activity, as the CSF–brain barrier is partially permeable. However, functional effects of CSF on neuronal network activity have not been specified to date. Here, we report that CSF specimens from patients with reduced Glasgow Coma Scale values caused by severe traumatic brain injury suppress synchronous activity of in vitro-generated neuronal networks in comparison with controls. We present evidence that underlying mechanisms include increased N-methyl-D-aspartate receptor activity mediated by a CSF fraction containing elevated amino acid concentrations. These proof-of-principle data suggest that determining effects of CSF specimens on neuronal network activity might be of diagnostic value. Ann Neurol 2009;66:546–555

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Sebastian Illes, Stephan Theiss, Hans-Peter Hartung, Mario Siebler, and Marcel Dihné

Abstract

Background

The present work was performed to investigate the ability of two different embryonic stem (ES) cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs), progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far.

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Sebastian Illes, Wiebke Fleischer, Mario Siebler, Hans-Peter Hartung, Marcel Dihné

Abstract

Embryonic stem cells can be differentiated into neurons of diverse neurotransmitter-specific phenotypes. While the time course of functional progression of ES cell-derived neural precursors towards mature neurons has been described in detail on single-cell level, the temporal development and pharmacological modulation of ES cell-derived neuronal network activity have not been explored yet. Neuronal network activity can be assessed by the microelectrode array (MEA) technology that allows simultaneous monitoring of the electrical activity exhibited by entire populations of neurons over several weeks or months in vitro.

We demonstrate here that ES cell-derived neural precursors cultured on MEAs for 5 to 6 weeks develop neuronal networks with oscillating and synchronous spike patterns via distinct states of activity and change electrophysiological characteristics even after 5 to 6 weeks in culture pointing towards late maturational processes. These processes were accompanied by an increasing density of presynaptic vesicles. Furthermore, we demonstrated that ES cell-derived network activity was sensitive to synaptically acting drugs indicating that pharmacologically susceptible neuronal networks were generated. Thus, the MEA technology represents a powerful tool to describe the temporal progression of stem cell-derived neural populations towards mature, functioning neuronal networks that can be applied to investigate pharmacologically active compounds.

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