Multielectrode Array (MEA)-Based Detection of Spontaneous Network Activity in Human iPSC-Derived Cortical Neurons

Abstract Background Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72RE) mutation – the most common genetic impairment causal to ALS and FTD. Noting that perturbations in cortical function are evidenced pre-symptomatically, and that the cortex is associated with widespread pathology, cortical dysfunction is thought to be an early driver of neurodegenerative disease progression. However, our understanding of how altered network function manifests at the cellular and molecular level is not clear. Methods To address this we have generated cortical neurons from patient-derived iPSCs harbouring C9ORF72RE mutations, as well as from their isogenic expansion-corrected controls. We have established a model of network activity in these neurons using multi-electrode array electrophysiology. We have then mechanistically examined the physiological processes underpinning network dysfunction using a combination of patch-clamp electrophysiology, immunocytochemistry, pharmacology and transcriptomic profiling. Results We find that C9ORF72RE causes elevated network burst activity, associated with enhanced synaptic input, yet lower burst duration, attributable to impaired pre-synaptic vesicle dynamics. We also show that the C9ORF72RE is associated with impaired synaptic plasticity. Moreover, RNA-seq analysis revealed dysregulated molecular pathways impacting on synaptic function. All molecular, cellular and network deficits are rescued by CRISPR/Cas9 correction of C9ORF72RE. Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD. Conclusion These findings suggest synaptic pathophysiology is widespread in ALS-FTD and has an early and fundamental role in driving altered network function that is thought to contribute to neurodegenerative processes in these patients. The overall importance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic plasticity, synaptic vesicle stores, and network propagation, which directly impact upon cortical function.

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Aldolase C/Zebrin II is Released to the Extracellular Space after Stroke and Inhibits the Network Activity of Cortical Neurons

Neurochemical Research ◽

10.1007/s11064-006-9169-9 ◽

2006 ◽

Vol 31 (11) ◽

pp. 1297-1303 ◽

Cited By ~ 10

Author(s):

Stephanie Linke ◽

Philipp Goertz ◽

Stephan L. Baader ◽

Volkmar Gieselmann ◽

Mario Siebler ◽

...

Keyword(s):

Cortical Neurons ◽

Extracellular Space ◽

Network Activity ◽

Aldolase C ◽

Zebrin Ii

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Acute Stimulation of Dissociated Cortical Neurons of Newborn Rats with Orexin A: Effect on the Network Activity

IFMBE Proceedings - 8th International Conference on Cell & Stem Cell Engineering (ICCE) ◽

10.1007/978-3-642-19044-5_10 ◽

2011 ◽

pp. 35-38 ◽

Cited By ~ 1

Author(s):

I. I. Stoyanova ◽

J. Feber ◽

W. L. C. Rutten

Keyword(s):

Cortical Neurons ◽

Network Activity ◽

Newborn Rats ◽

Orexin A ◽

Dissociated Cortical Neurons ◽

Stimulation Of

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Spontaneous dynamics of neural networks in deep layers of prefrontal cortex

Journal of Neurophysiology ◽

10.1152/jn.00295.2016 ◽

2017 ◽

Vol 117 (4) ◽

pp. 1581-1594 ◽

Cited By ~ 4

Author(s):

Andrew S. Blaeser ◽

Barry W. Connors ◽

Arto V. Nurmikko

Keyword(s):

Prefrontal Cortex ◽

Spontaneous Activity ◽

Calcium Imaging ◽

Cortical Neurons ◽

Rhythmic Activity ◽

Network Activity ◽

Local Networks ◽

Spatiotemporal Scale ◽

Deep Layers ◽

Delta Oscillations

Cortical systems maintain and process information through the sustained activation of recurrent local networks of neurons. Layer 5 is known to have a major role in generating the recurrent activation associated with these functions, but relatively little is known about its intrinsic dynamics at the mesoscopic level of large numbers of neighboring neurons. Using calcium imaging, we measured the spontaneous activity of networks of deep-layer medial prefrontal cortical neurons in an acute slice model. Inferring the simultaneous activity of tens of neighboring neurons, we found that while the majority showed only sporadic activity, a subset of neurons engaged in sustained delta frequency rhythmic activity. Spontaneous activity under baseline conditions was weakly correlated between pairs of neurons, and rhythmic neurons showed little coherence in their oscillations. However, we consistently observed brief bouts of highly synchronous activity that must be attributed to network activity. NMDA-mediated stimulation enhanced rhythmicity, synchrony, and correlation within these local networks. These results characterize spontaneous prefrontal activity at a previously unexplored spatiotemporal scale and suggest that medial prefrontal cortex can act as an intrinsic generator of delta oscillations. NEW & NOTEWORTHY Using calcium imaging and a novel analytic framework, we characterized the spontaneous and NMDA-evoked activity of layer 5 prefrontal cortex at a largely unexplored spatiotemporal scale. Our results suggest that the mPFC microcircuitry is capable of intrinsically generating delta oscillations and sustaining synchronized network activity that is potentially relevant for understanding its contribution to cognitive processes.

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Variant-specific changes in persistent or resurgent Na+ current in SCN8A-EIEE13 iPSC-derived neurons

10.1101/2020.01.16.909192 ◽

2020 ◽

Author(s):

Andrew M. Tidball ◽

Luis F. Lopez-Santiago ◽

Yukun Yuan ◽

Trevor W. Glenn ◽

Joshua L. Margolis ◽

...

Keyword(s):

Cortical Neurons ◽

Disease Modeling ◽

Wide Spectrum ◽

Sodium Current ◽

Specific Model ◽

Epileptic Encephalopathy ◽

Network Activity ◽

Patient Specific ◽

Unexpected Death ◽

Missense Variants

AbstractMissense variants in the voltage-gated sodium channel (VGSC) gene, SCN8A, are linked to early-infantile epileptic encephalopathy type 13 (EIEE13). EIEE13 patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy (SUDEP). The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current (INa) properties. To investigate these mechanisms using a patient-specific model system, we generated induced pluripotent stem cells (iPSCs) from three patients with missense variants in SCN8A: p.R1872>L (P1); p.V1592>L (P2); and p.N1759>S (P3). Using small molecule differentiation into excitatory neurons, iPSC-derived neurons from all three patients displayed altered INa. P1 and P2 had elevated persistent INa, while P3 had increased resurgent INa compared to controls. Further analyses focused on one of the patients with increased persistent INa (P1) and the patient with increased resurgent INa (P3). Excitatory cortical neurons from both patients had prolonged action potential (AP) repolarization and shorter axon initial segment lengths compared to controls, the latter analyzed by immunostaining for ankyrin-G. Using doxycycline-inducible expression of the neuronal transcription factors Neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons (iNeurons), we investigated network activity and response to pharmacotherapies. Both patient neurons and iNeurons displayed similar abnormalities in AP repolarization. Patient iNeurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for EIEE patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent INa, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the AP firing threshold of patient-derived neurons to more depolarized potentials. Our results indicate that patient-specific neurons are useful for modeling EIEE13 and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific iPSC neuronal disease modeling offers a useful platform for discovering precision epilepsy therapies.

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Coordinated regulation of CB1 cannabinoid receptors and anandamide metabolism stabilize network activity during homeostatic scaling down

10.1101/2021.05.21.445170 ◽

2021 ◽

Author(s):

Michael Ye ◽

Sarah K Monroe ◽

Sean M Gay ◽

Michael L Armstrong ◽

Diane E Youngstrom ◽

...

Keyword(s):

Cell Surface ◽

Cortical Neurons ◽

Cannabinoid Receptors ◽

Acid Amide ◽

Synaptic Function ◽

Synaptic Activity ◽

Network Activity ◽

Bioactive Lipids ◽

Network Properties ◽

Scaling Down

Neurons express overlapping homeostatic mechanisms to regulate synaptic function and network properties in response to perturbations of neuronal activity. Endocannabinoids (eCBs) are bioactive lipids synthesized in the post-synaptic compartments that regulate synaptic transmission, plasticity, and neuronal excitability throughout much of the brain, by activating pre-synaptic cannabinoid receptor CB1. The eCB system is well situated to regulate neuronal network properties and coordinate pre- and post-synaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. We show that in mature cultured rat cortical neurons, chronic bicuculline treatment, known to induce homeostatic scaling-down, induces a coordinated adaptation to enhance tonic eCB signaling. Hyper-excitation triggers down regulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide. Subsequently, we measured an accumulation of anandamide and related metabolites, and an upregulation of total and cell surface CB1. We show that bicuculline induced downregulation of surface AMPA-type glutamate receptors and upregulation of CB1 occur through independent mechanisms. Finally, using live-cell microscopy of neurons expressing an extracellular fluorescent glutamate reporter (iGluSnFR), we confirm that cortical neurons in vitro exhibit highly synchronized network activity, reminiscent of cortical up-states in vivo. Up-state like activity in mature cortical cultures requires CB1 signaling under both control conditions and following chronic bicuculline treatment. We propose that during the adaptation to chronic neuronal hyperexcitation, tonic eCB signaling is enhanced through coordinated changes in anandamide metabolism and cell-surface CB1 expression to maintain synchronous network activity.

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Network activity influences the subthreshold and spiking visual responses of pyramidal neurons in a three-layer cortex

10.1101/087288 ◽

2016 ◽

Author(s):

Nathaniel C. Wright ◽

Ralf Wessel

Keyword(s):

Membrane Potential ◽

Cortical Neurons ◽

Visual Stimulation ◽

Ex Vivo ◽

Network Activity ◽

Visual Responses ◽

Cortical Function ◽

Sensory Evoked ◽

High Conductance

A primary goal of systems neuroscience is to understand cortical function, which typically involves studying spontaneous and sensory-evoked cortical activity. Mounting evidence suggests a strong and complex relationship between the ongoing and evoked state. To date, most work in this area has been based on spiking in populations of neurons. While advantageous in many respects, this approach is limited in scope; it records the activities of a minority of neurons, and gives no direct indication of the underlying subthreshold dynamics. Membrane potential recordings can fill these gaps in our understanding, but are difficult to obtain in vivo. Here, we record subthreshold cortical visual responses in the ex vivo turtle eye-attached whole-brain preparation, which is ideally-suited to such a study. In the absence of visual stimulation, the network is “synchronous”; neurons display network-mediated transitions between low- and high-conductance membrane potential states. The prevalence of these slow-wave transitions varies across turtles and recording sessions. Visual stimulation evokes similar high-conductance states, which are on average larger and less reliable when the ongoing state is more synchronous. Responses are muted when immediately preceded by large, spontaneous high-conductance events. Evoked spiking is sparse, highly variable across trials, and mediated by concerted synaptic inputs that are in general only very weakly correlated with inputs to nearby neurons. Together, these results highlight the multiplexed influence of the cortical network on the spontaneous and sensory-evoked activity of individual cortical neurons.

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KCC2 overexpression prevents the paradoxical seizure-promoting action of somatic inhibition

10.1101/279539 ◽

2018 ◽

Cited By ~ 1

Author(s):

Vincent Magloire ◽

Jonathan Cornford ◽

Andreas Lieb ◽

Dimitri M. Kullmann ◽

Ivan Pavlov

Keyword(s):

Potassium Chloride ◽

Cortical Neurons ◽

Closed Loop ◽

Network Activity ◽

Intracellular Chloride ◽

Cortical Interneurons ◽

The Face ◽

Somatic Inhibition

AbstractAlthough cortical interneurons are apparently well-placed to suppress seizures, several recent reports have highlighted a paradoxical role of parvalbumin-positive perisomatic-targeting (PV+) interneurons in ictogenesis. Here, we use an acute in vivo model of focal cortical seizures in awake behaving mice, together with closed-loop optogenetic manipulation of PV+ interneurons, to investigate their function during seizures. We show that photo-depolarization of PV+ interneurons rapidly switches from an anti-ictal to a pro-ictal effect within a few seconds of seizure initiation. The pro-ictal effect of delayed photostimulation of PV+ interneurons was not shared with dendrite-targeting somatostatin-positive (SOM+) interneurons. We also show that this switch can be prevented by overexpression of the neuronal potassium-chloride co-transporter KCC2 in principal cortical neurons. These results suggest that strategies aimed at improving the ability of principal neurons to maintain intracellular chloride levels in the face of excessive network activity can prevent interneurons from contributing to seizure perpetuation.

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

Translational Psychiatry ◽

10.1038/s41398-021-01399-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Julia Izsak ◽

Henrik Seth ◽

Margarita Iljin ◽

Stephan Theiss ◽

Hans Ågren ◽

...

Keyword(s):

Patch Clamp ◽

Receptor Antagonist ◽

Single Cell ◽

Ampa Receptor ◽

Neuronal Network ◽

Cortical Neurons ◽

Network Activity ◽

Lithium Salts ◽

Neuronal Function ◽

Neuronal Network Activity

AbstractLithium 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.

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