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In this article, we aim to study how astrocytes control electrophysiological activity during neuronal network formation. We used a combination of spike/burst analysis, classification of spike waveforms based on various signal properties and tools from information theory to demonstrate how astrocytes modulate the electrical activity of neurons using microelectrode array (MEA) signals. We cultured rat primary cortical neurons and astrocytes on 60-electrode MEAs with different neuron/astrocyte ratios ranging from 'pure' neuronal cultures to co-cultures containing 50% neurons and 50% astrocytes. Our results show that astrocytes desynchronize the network and reduce predictability in the signals and affect the repolarization phase of the action potentials. Our work highlights that it is crucial to go beyond standard MEA analysis to assess how astrocytes control neuronal cultures and investigate any dysfunction that could potentially result in neuronal hyperactivity., Competing Interests: We declare we have no competing interests., (© 2024 The Author(s).)

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain that binds to GABA receptors and hyperpolarizes the postsynaptic neuron. Gabazine acts as a competitive antagonist to type A GABA receptors (GABA A R), thereby causing diminished neuronal hyperpolarization and GABA A R-mediated inhibition. However, the biochemical effects and the potential regulatory role of astrocytes in this process remain poorly understood. To address this, we investigated the neuronal responses of gabazine in rat cortical cultures containing varying ratios of neurons and astrocytes. Electrophysiological characterization was performed utilizing microelectrode arrays (MEAs) with topologically controlled microcircuit cultures that enabled control of neuronal network growth. Biochemical analysis of the cultures was performed using traditional dissociated cultures on coverslips. Our study indicates that, upon gabazine stimulation, astrocyte-rich neuronal cultures exhibit elevated electrophysiological activity and tyrosine phosphorylation of tropomyosin receptor kinase B (TrkB; receptor for brain-derived neurotrophic factor), along with distinct cytokine secretion profiles. Notably, neurons lacking proper astrocytic support were found to experience synapse loss and decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, astrocytes contributed to neuronal viability, morphology, vascular endothelial growth factor (VEGF) secretion, and overall neuronal network functionality, highlighting the multifunctional role of astrocytes., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Bottom-up neuroscience utilizes small, engineered biological neural networks to study neuronal activity in systems of reduced complexity. We present a platform that establishes up to six independent networks formed by primary rat neurons on planar complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs). We introduce an approach that allows repetitive stimulation and recording of network activity at any of the over 700 electrodes underlying a network. We demonstrate that the continuous application of a repetitive super-threshold stimulus yields a reproducible network answer within a 15 ms post-stimulus window. This response can be tracked with high spatiotemporal resolution across the whole extent of the network. Moreover, we show that the location of the stimulation plays a significant role in the networks' early response to the stimulus. By applying a stimulation pattern to all network-underlying electrodes in sequence, the sensitivity of the whole network to the stimulus can be visualized. We demonstrate that microchannels reduce the voltage stimulation threshold and induce the strongest network response. By varying the stimulation amplitude and frequency we reveal discrete network transition points. Finally, we introduce vector fields to follow stimulation-induced spike propagation pathways within the network. Overall we show that our defined neural networks on CMOS MEAs enable us to elicit highly reproducible activity patterns that can be precisely modulated by stimulation amplitude, stimulation frequency and the site of stimulation., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Astrocytes and neurons respond to each other by releasing transmitters, such as γ-aminobutyric acid (GABA) and glutamate, that modulate the synaptic transmission and electrochemical behavior of both cell types. Astrocytes also maintain neuronal homeostasis by clearing neurotransmitters from the extracellular space. These astrocytic actions are altered in diseases involving malfunction of neurons, e.g., in epilepsy, Alzheimer's disease, and Parkinson's disease. Convulsant drugs such as 4-aminopyridine (4-AP) and gabazine are commonly used to study epilepsy in vitro. In this study, we aim to assess the modulatory roles of astrocytes during epileptic-like conditions and in compensating drug-elicited hyperactivity. We plated rat cortical neurons and astrocytes with different ratios on microelectrode arrays, induced seizures with 4-AP and gabazine, and recorded the evoked neuronal activity. Our results indicated that astrocytes effectively counteracted the effect of 4-AP during stimulation. Gabazine, instead, induced neuronal hyperactivity and synchronicity in all cultures. Furthermore, our results showed that the response time to the drugs increased with an increasing number of astrocytes in the co-cultures. To the best of our knowledge, our study is the first that shows the critical modulatory role of astrocytes in 4-AP and gabazine-induced discharges and highlights the importance of considering different proportions of cells in the cultures.

Cortical spreading depression (CSD) is a slowly propagating wave of depolarization of brain cells, followed by temporary silenced electrical brain activity. Major structural changes during CSD are linked to neuronal and possibly glial swelling. However, basic questions still remain unanswered. In particular, there are open questions regarding whether neurons or glial cells swell more, and how the cellular swelling affects the CSD wave propagation.In this study, we computationally explore how different parameters affect the swelling of neurons and astrocytes (starshaped glial cells) during CSD and how the cell swelling alters the CSD wave spatial distribution. We apply a homogenized mathematical model that describes electrodiffusion in the intraand extracellular space, and discretize the equations using a finite element method. The simulations are run with a twocompartment (extracellular space and neurons) and a threecompartment version of the model with astrocytes added. We consider cell swelling during CSD in four scenarios: (A) incorporating aquaporin-4 channels in the astrocytic membrane, (B) increasing the neuron/astrocyte ratio to 2:1, (C) blocking and increasing the Na + /K + -ATPase rate in the astrocytic compartment, and (D) blocking the Cl - channels in astrocytes. Our results show that increasing the water permeability in the astrocytes results in a higher astrocytic swelling and a lower neuronal swelling than in the default case. Further, elevated neuronal density increases the swelling in both neurons and astrocytes. Blocking the Na + /K + -ATPase in the astrocytes leads to an increased wave width and swelling in both compartments, which instead decreases when the pump rate is raised. Blocking the Cl - channels in the astrocytes results in neuronal swelling, and a shrinkage in the astrocytes. Our results suggest a supporting role of astrocytes in preventing cellular swelling and CSD, as well as highlighting how dysfunctions in astrocytes might elicit CSD.