RESUMO
Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here, we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture. Whole-cell patch-clamp demonstrated subthreshold synaptic inputs and action potential firing of neurons. Spontaneous intracellular calcium signals turned into synchronous large-scale oscillations upon combined disinhibition of NMDA receptors and blocking of GABAA receptors. Brief metabolic inhibition to mimic transient energy restriction in the ischemic brain induced reversible intracellular calcium loading of cBOS. Moreover, metabolic inhibition induced a reversible decline in neuronal ATP as revealed by ATeam1.03YEMK. Overall, cBOS provide a powerful platform to assess morphological and functional aspects of human neural cells in intact minimal networks and to address the pathways that drive cellular damage during brain ischemia.
RESUMO
KEY POINTS: Employing quantitative Na+ -imaging and Förster resonance energy transfer-based imaging with ATeam1.03YEMK (ATeam), we studied the relation between activity-induced Na+ influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus. Calibration of ATeam in situ enabled a quantitative estimate of changes in intracellular ATP concentrations. Different paradigms of stimulation that induced global Na+ influx into the entire neuron resulted in decreases in [ATP] in the range of 0.1-0.6 mm in somata and dendrites, while Na+ influx that was locally restricted to parts of dendrites did not evoke a detectable change in dendritic [ATP]. Our data suggest that global Na+ transients require global cellular activation of the Na+ /K+ -ATPase resulting in a consumption of ATP that transiently overrides its production. For recovery from locally restricted Na+ influx, ATP production as well as fast intracellular diffusion of ATP and Na+ might prevent a local drop in [ATP]. ABSTRACT: Excitatory neuronal activity results in the influx of Na+ through voltage- and ligand-gated channels. Recovery from accompanying increases in intracellular Na+ concentrations ([Na+ ]i ) is mainly mediated by the Na+ /K+ -ATPase (NKA) and is one of the major energy-consuming processes in the brain. Here, we analysed the relation between different patterns of activity-induced [Na+ ]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na+ imaging with sodium-binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i ) with an apparent KD of 2.6 mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na+ ]i oscillations with amplitudes of â¼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3-0.4 mm in both compartments. Global [Na+ ]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na+ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i . Our results suggest that ATP consumption by the NKA following global [Na+ ]i transients temporarily overrides its availability, causing a decrease in [ATP]i . Locally restricted Na+ transients, however, do not result in detectable changes in local [ATP]i , suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na+ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions.