Differential acute impact of therapeutically effective and overdose concentrations of lithium on human neuronal single cell and network function.

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 GABAA-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.

Abnormal gene expression of BDNF, but not BDNF-AS, in iPSC, neural stem cells and postmortem brain samples from bipolar disorder.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) antisense RNA (BDNF-AS) was identified as naturally conserved non-coding antisense RNA that suppresses the transcription of BDNF. METHODS: We measured the expression of BDNF mRNA and BDNF-AS mRNA in iPSC and NSC from bipolar disorder (BD) patients and healthy control subjects, and postmortem brain samples such as the corpus callosum, the Brodmann area (BA8), and BA46 from BD patients and age- and sex-matched controls. RESULTS: The expression of BDNF mRNA in iPSC from BD patients (n = 6) was significantly lower than that of control subjects (n = 4) although the expression of BDNF mRNA in NSC from BD patients was significantly higher than that of control subjects. In contrast, there were no changes in the expression of BDNF-AS mRNA in both iPSC and NSC between two groups. The expression of BDNF mRNA in the BA46 from BD patients (n = 35) was significantly lower than that of controls (n = 34) although the expression of BDNF mRNA in the corpus callosum and BA8 was not different between two groups (n = 15). In contrast, there were no changes in expression of BDNF-AS mRNA in the three brain regions between two groups. Interestingly, there were significant positive correlations between BDNF mRNA expression and BDNF-AS mRNA expression in the postmortem brain samples. LIMITATIONS: Sample sizes are relatively low. CONCLUSIONS: Our data suggest that abnormalities in the expression of BDNF, but not BDNF-AS, play a role in the pathogenesis of BD.

TGF-ß1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural in vitro Models.

Persistent neural stem cell (NSC) proliferation is, among others, a hallmark of immaturity in human induced pluripotent stem cell (hiPSC)-based neural models. TGF-ß1 is known to regulate NSCs in vivo during embryonic development in rodents. Here we examined the role of TGF-ß1 as a potential candidate to promote in vitro differentiation of hiPSCs-derived NSCs and maturation of neuronal progenies. We present that TGF-ß1 is specifically present in early phases of human fetal brain development. We applied confocal imaging and electrophysiological assessment in hiPSC-NSC and 3D neural in vitro models and demonstrate that TGF-ß1 is a signaling protein, which specifically suppresses proliferation, enhances neuronal and glial differentiation, without effecting neuronal maturation. Moreover, we demonstrate that TGF-ß1 is equally efficient in enhancing neuronal differentiation of human NSCs as an artificial synthetic small molecule. The presented approach provides a proof-of-concept to replace artificial small molecules with more physiological signaling factors, which paves the way to improve the physiological relevance of human neural developmental in vitro models.

Human Cerebrospinal Fluid Promotes Neuronal Circuit Maturation of Human Induced Pluripotent Stem Cell-Derived 3D Neural Aggregates.

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.

Partial reduction of amyloid ß production by ß-secretase inhibitors does not decrease synaptic transmission.

BACKGROUND: Alzheimer's disease (AD) is the most common form of age-related neurodegenerative diseases. Cerebral deposition of Aß peptides, especially Aß42, is considered the major neuropathological hallmark of AD and the putative cause of AD-related neurotoxicity. Aß peptides are produced by sequential proteolytic processing of APP, with ß-secretase (BACE) being the initiating enzyme. Therefore, BACE has been considered an attractive therapeutic target in AD research and several BACE inhibitors have been tested in clinical trials, but so far, all have had negative outcomes or even led to worsening of cognitive function. AD can be triggered by Aß years before the first symptoms appear and one reason for the failures could be that the clinical trials were initiated too late in the disease process. Another possible explanation could be that BACE inhibition alters physiological APP processing in a manner that impairs synaptic function, causing cognitive deterioration. METHODS: The aim of this study was to investigate if partial BACE inhibition, mimicking the putative protective effect of the Icelandic mutation in the APP gene, could reduce Aß generation without affecting synaptic transmission. To investigate this, we used an optical electrophysiology platform, in which effects of compounds on synaptic transmission in cultured neurons can be monitored. We employed this method on primary cortical rat neuronal cultures treated with three different BACE inhibitors (BACE inhibitor IV, LY2886721, and lanabecestat) and monitored Aß secretion into the cell media. RESULTS: We found that all three BACE inhibitors tested decreased synaptic transmission at concentrations leading to significantly reduced Aß secretion. However, low-dose BACE inhibition, resulting in less than a 50% decrease in Aß secretion, did not affect synaptic transmission for any of the inhibitors tested. CONCLUSION: Our results indicate that Aß production can be reduced by up to 50%, a level of reduction of relevance to the protective effect of the Icelandic mutation, without causing synaptic dysfunction. We therefore suggest that future clinical trials aimed at prevention of Aß build-up in the brain should aim for a moderate CNS exposure of BACE inhibitors to avoid side effects on synaptic function.

Robust Generation of Person-Specific, Synchronously Active Neuronal Networks Using Purely Isogenic Human iPSC-3D Neural Aggregate Cultures.

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.

Neuromodulation via the Cerebrospinal Fluid: Insights from Recent in Vitro Studies.

The cerebrospinal fluid (CSF) occupies the brain's ventricles and subarachnoid space and, together with the interstitial fluid (ISF), forms a continuous fluidic network that bathes all cells of the central nervous system (CNS). As such, the CSF is well positioned to actively distribute neuromodulators to neural circuits in vivo via volume transmission. Recent in vitro experimental work in brain slices and neuronal cultures has shown that human CSF indeed contains neuromodulators that strongly influence neuronal activity. Here we briefly summarize these new findings and discuss their potential relevance to neural circuits in health and disease.

More than a drainage fluid: the role of CSF in signaling in the brain and other effects on brain tissue.

Current progress in neuroscience demonstrates that the brain is not an isolated organ and is influenced by the systemic environment and extracerebral processes within the body. In view of this new concept, blood and cerebrospinal fluid (CSF) are important body fluids linking extracerebral and intracerebral processes. For decades, substantial evidence has been accumulated indicating that CSF modulates brain states and influences behavior as well as cognition. This chapter provides an overview of how CSF directly modulates the function of different types of brain cells, such as neurons, neural stem cells, and CSF-contacting cells. Alterations in CSF content occur in most pathologic central nervous system (CNS) conditions. In a classic view, the function of CSF is to drain waste products and detrimental factors derived from diseased brain parenchyma. This chapter presents examples for how intra- and extracerebral pathologic processes lead to alterations in the CSF content. Current knowledge about how pathologically altered CSF influences the functionality of brain cells will be presented. Thereby, it becomes evident that CSF has more than a drainage function and has a causal role for the etiology and pathogenesis of different CNS diseases.

Analysis and Modeling of Subthreshold Neural Multi-Electrode Array Data by Statistical Field Theory.

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.

Corrigendum: Human Cerebrospinal Fluid Promotes Neuronal Viability and Activity of Hippocampal Neuronal Circuits In Vitro.

[This corrects the article on p. 54 in vol. 10, PMID: 26973467.].

Human Cerebrospinal Fluid Promotes Neuronal Viability and Activity of Hippocampal Neuronal Circuits In Vitro.

For decades it has been hypothesized that molecules within the cerebrospinal fluid (CSF) diffuse into the brain parenchyma and influence the function of neurons. However, the functional consequences of CSF on neuronal circuits are largely unexplored and unknown. A major reason for this is the absence of appropriate neuronal in vitro model systems, and it is uncertain if neurons cultured in pure CSF survive and preserve electrophysiological functionality in vitro. In this article, we present an approach to address how human CSF (hCSF) influences neuronal circuits in vitro. We validate our approach by comparing the morphology, viability, and electrophysiological function of single neurons and at the network level in rat organotypic slice and primary neuronal cultures cultivated either in hCSF or in defined standard culture media. Our results demonstrate that rodent hippocampal slices and primary neurons cultured in hCSF maintain neuronal morphology and preserve synaptic transmission. Importantly, we show that hCSF increases neuronal viability and the number of electrophysiologically active neurons in comparison to the culture media. In summary, our data indicate that hCSF represents a physiological environment for neurons in vitro and a superior culture condition compared to the defined standard media. Moreover, this experimental approach paves the way to assess the functional consequences of CSF on neuronal circuits as well as suggesting a novel strategy for central nervous system (CNS) disease modeling.

Structural and functional rejuvenation of the aged brain by an approved anti-asthmatic drug.

As human life expectancy has improved rapidly in industrialized societies, age-related cognitive impairment presents an increasing challenge. Targeting histopathological processes that correlate with age-related cognitive declines, such as neuroinflammation, low levels of neurogenesis, disrupted blood-brain barrier and altered neuronal activity, might lead to structural and functional rejuvenation of the aged brain. Here we show that a 6-week treatment of young (4 months) and old (20 months) rats with montelukast, a marketed anti-asthmatic drug antagonizing leukotriene receptors, reduces neuroinflammation, elevates hippocampal neurogenesis and improves learning and memory in old animals. By using gene knockdown and knockout approaches, we demonstrate that the effect is mediated through inhibition of the GPR17 receptor. This work illustrates that inhibition of leukotriene receptor signalling might represent a safe and druggable target to restore cognitive functions in old individuals and paves the way for future clinical translation of leukotriene receptor inhibition for the treatment of dementias.

Intrinsically active and pacemaker neurons in pluripotent stem cell-derived neuronal populations.

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.

In vitro neuronal network activity in NMDA receptor encephalitis.

BACKGROUND: Anti-NMDA-encephalitis is caused by antibodies against the N-methyl-D-aspartate receptor (NMDAR) and characterized by a severe encephalopathy with psychosis, epileptic seizures and autonomic disturbances. It predominantly occurs in young women and is associated in 59% with an ovarian teratoma. RESULTS: We describe effects of cerebrospinal fluid (CSF) from an anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis patient on in vitro neuronal network activity (ivNNA). In vitro NNA of dissociated primary rat cortical populations was recorded by the microelectrode array (MEA) system.The 23-year old patient was severely affected but showed an excellent recovery following multimodal immunomodulatory therapy and removal of an ovarian teratoma. Patient CSF (pCSF) taken during the initial weeks after disease onset suppressed global spike- and burst rates of ivNNA in contrast to pCSF sampled after clinical recovery and decrease of NMDAR antibody titers. The synchrony of pCSF-affected ivNNA remained unaltered during the course of the disease. CONCLUSION: Patient CSF directly suppresses global activity of neuronal networks recorded by the MEA system. In contrast, pCSF did not regulate the synchrony of ivNNA suggesting that NMDAR antibodies selectively regulate distinct parameters of ivNNA while sparing their functional connectivity. Thus, assessing ivNNA could represent a new technique to evaluate functional consequences of autoimmune encephalitis-related CSF changes.

A new role for interferon gamma in neural stem/precursor cell dysregulation.

BACKGROUND: The identification of factors that compromise neurogenesis is aimed at improving stem cell-based approaches in the field of regenerative medicine. Interferon gamma (IFNγ) is a main pro-inflammatory cytokine and up-regulated during several neurological diseases. IFNγ is generally thought to beneficially enhance neurogenesis from fetal or adult neural stem/precursor cells (NSPCs). RESULTS: We now provide direct evidence to the contrary that IFNγ induces a dysfunctional stage in a substantial portion of NSPC-derived progeny in vitro characterized by simultaneous expression of glial fibrillary acid protein (GFAP) and neuronal markers, an abnormal gene expression and a functional phenotype neither typical for neurons nor for mature astrocytes. Dysfunctional development of NSPCs under the influence of IFNγ was finally demonstrated by applying the microelectrode array technology. IFNγ exposure of NSPCs during an initial 7-day proliferation period prevented the subsequent adequate differentiation and formation of functional neuronal networks. CONCLUSIONS: Our results show that immunocytochemical analyses of NSPC-derived progeny are not necessarily indicating the correct cellular phenotype specifically under inflammatory conditions and that simultaneous expression of neuronal and glial markers rather point to cellular dysregulation. We hypothesize that inhibiting the impact of IFNγ on NSPCs during neurological diseases might contribute to effective neurogenesis and regeneration.

Cerebrospinal fluid of brain trauma patients inhibits in vitro neuronal network function via NMDA receptors.

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.

Protein tyrosine phosphatases expression during development of mouse superior colliculus.

Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis.

Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations.

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. RESULTS: While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2), only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 24 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated vGlut2 expression within presynaptic vesicles. Also those NPCs that had migrated away from adherent neural aggregates maintained their ability to generate a synchronously oscillating neuronal network, even if they were separated from adherent aggregates, dissociated and re-plated. CONCLUSION: These findings suggest that the complex environment within niches and aggregates of heterogeneous neural cell populations support the generation of fully mature neurons and functional neuronal networks from ES cell-derived neural cells. In contrast, homogeneous ES cell-derived NPCs within monolayer cultures exhibited an impaired functional neuronal maturation.

Development and pharmacological modulation of embryonic stem cell-derived neuronal network activity.

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.