Human brain assembloids as a model of anaesthetic-induced neural dynamics in vitro

The mechanisms by which anaesthetics alter neurophysiological states remain poorly understood. Although some hypotheses emphasise thalamic suppression, others propose that cortical (Cx) circuits alone, comprising interconnected excitatory and inhibitory neurones expressing gamma-aminobutyric acid type A (GABA_A) receptors, are sufficient for anaesthetic-induced neural dynamics.

We generated human induced pluripotent stem cell-derived Cx-ganglionic eminence (GE) assembloids and characterised their cellular composition using immunohistochemistry and single-nucleus RNA sequencing. We recorded local field potentials and microelectrode array activity from assembloids exposed to 96 μM propofol, analysing changes in extracellular field delta power (0.1-4 Hz) and Lempel-Ziv complexity, and neuronal firing rates. Control experiments included propofol application to excitatory Cx-only organoids and co-application of propofol with the competitive GABA_A receptor antagonist bicuculline.

Cx-GE assembloids expressed propofol-sensitive GABA_A receptor β2/β3 subunits on both excitatory and inhibitory neurones and chloride homeostasis machinery sufficient for hyperpolarising GABA responses. Propofol exposure significantly increased relative delta power (P=0.0078), reduced signal complexity (P=0.0117) of local field potentials, and suppressed neuronal firing rates (P=0.0156). These effects were absent in excitatory Cx-only organoids and were abolished by GABA_A receptor antagonism with bicuculline, confirming the requirement for both interneurones and GABAergic signalling. Assembloids also expressed genes for neuromodulatory receptors implicated in anaesthetic emergence.

Human Cx-GE assembloids recapitulate key electrophysiological features of anaesthetic states, demonstrating that a minimal Cx circuit containing excitatory and inhibitory neurones with functional GABA_A receptors is sufficient to generate anaesthetic-induced alterations in neural dynamics. This tractable human-derived model helps disentangle Cx from subcortical contributions to anaesthesia and establishes a potential in vitro platform for mechanistic studies of anaesthetic-induced neural dynamics, drug screening, and investigation of clinically relevant individual differences in anaesthetic sensitivity, including genetic risk factors for unintentional awareness with recall and perioperative neurocognitive disorders, without the confounds of intact subcortical circuitry or the ethical constraints of human experimentation.