Astrocytes facilitate gabazine‐evoked electrophysiological hyperactivity and distinct biochemical responses in mature neuronal cultures

• Published in: Journal of neurochemistry 2024-09, Vol.168 (9), p.3076-3094
• Main Authors: Ahtiainen, Annika, Genocchi, Barbara, Subramaniyam, Narayan Puthanmadam, Tanskanen, Jarno M. A., Rantamäki, Tomi, Hyttinen, Jari A. K.
• Format: Article
• Language: English
• Subjects: Astrocytes; Biochemical analysis; Biochemistry; Brain; co‐culture; gabazine; gamma‐aminobutyric acid; Growth factors; Hyperactivity; Hyperpolarization; Kinases; MAP kinase; microelectrode array; Microelectrodes; Neural networks; Neurons; Neurotrophic factors; Phosphorylation; Receptors; TrkB; TrkB receptors; Tropomyosin; Tyrosine; Vascular endothelial growth factor; γ-Aminobutyric acid A receptors; γ-Aminobutyric acid receptors
• ISSN: 0022-3042; 1471-4159; 1471-4159
• DOI: 10.1111/jnc.16182

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 (GABAAR), thereby causing diminished neuronal hyperpolarization and GABAAR‐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. This study investigated the effects of the GABAAR selective competitive antagonist gabazine on neural cell cultures. The cultures contained various ratios of neurons and astrocytes, examining the role of astrocytes in neuronal electrophysiology and biochemistry. The setup included a topologically‐controlled microcircuit system on microelectrode arrays for electrophysiological assessment and dissociated cell cultures on coverslips for biochemical analysis. Astrocytes modulated neuronal responses to gabazine, resulting in increased electrical activity in astrocyte‐rich co‐cultures. Moreover, co‐cultures exhibited distinct cytokine secretions, most notably the secretion of VEGF, improved viability, and enhanced neuronal network connectivity. Co‐cultures with astrocyte support had increased p‐TrkB levels and synapse preservation after gabazine administration. These findings shed light on the mechanisms by which a