Distinct effects of different metabolic stress models on human-derived neuronal networks

Different in vitro models are widely used as experimental platforms to assess neuronal responses to metabolic stress and test potential treatments for patients with ischemic stroke. Results of those studies depend on the stress models used, and the link between cell viability-based readouts and electrophysiological activity remains poorly explored. We investigated the neuronal network activity of human-derived neuronal networks generated from human induced pluripotent stem cells (hiPSCs) under three commonly used metabolic stress models: hypoxia alone, oxygen and glucose deprivation (OGD), and hypoxia combined with different concentrations of glutamate. We aim to clarify the differences between three commonly used in vitro models, including the relation between microscopic and electrophysiological readouts. These conditions produced distinct effects on neuronal network activity. Hypoxia alone induced a progressive decline in activity over time. In contrast, OGD triggered a biphasic response, characterized by an early increase in activity followed by a decline. High concentration glutamate exposure under hypoxia also altered network dynamics, inducing a triphasic pattern consisting of a rapid activity decrease, a transient increase, and a subsequent decline. Across all these pathological conditions, neuronal activity progressively declined and converged toward network failure after prolonged hypoxia. Following reoxygenation, recovery was limited and condition-dependent: hypoxia alone, OGD, and high glutamate conditions showed limited recovery. On the other hand, low glutamate concentration was associated with good recovery. Microscopic assessment revealed that cellular viability was differentially affected across conditions. OGD was associated with the highest levels of cell death, whereas glutamate exposure, particularly at high concentrations, led to a marked reduction in synaptic puncta despite partial preservation of cell viability. These findings highlight that commonly used in vitro ischemia models induce distinct neuronal responses and highlight the importance of integrating electrophysiological and structural analyses to better characterize metabolic stress in human neuronal networks better.