Characterising the anxiogenic network from functional connectivity analysis of the CO₂ challenge model

The CO₂ challenge model (CCM) is a gas inhalation paradigm that provides precisely controlled anxiety induction in experimental settings. Despite its potential as an experimental model of anxiety, our understanding of the neural effects of the CCM is incomplete. This study employs resting-state functional magnetic resonance imaging (rs-fMRI) to explore functional connectivity (FC) changes underlying the CCM. Following a preliminary CO₂ tolerance assessment, participants completed an MRI session that included three rs-fMRI scans: during inhalation of control air (pre and post), and during a 6% CCM exposure. Here, we confirm that 6% CCM is a tolerable anxiogenic model in the MRI setting. We demonstrate that a transient CCM-induced increase in subjective anxiety is associated with an increase in FC within limbic and anxiety-related regions, with the insula emerging as a central node in this altered connectivity pattern. Further analysis revealed a significant correlation between the levels of subjective anxiety and enhanced FC between the brainstem and medial frontal cortex, highlighting the dynamic role of the brainstem in response to CO₂-induced anxiety. These findings underscore the value of combining CCM and rs-fMRI to characterise the neural mechanisms of anxiety, with important implications for evaluating potential therapeutic interventions.