Cortical Silencing Results in Paradoxical fMRI Overconnectivity

fMRI‐based measurements of functional connectivity are commonly interpreted as an index of anatomical coupling and direct interareal communication. However, causal testing of this hypothesis has been lacking. Here we combine neural silencing, resting‐state fMRI and in vivo electrophysiology to causally probe how inactivation of a cortical region affects brain‐wide functional coupling. We find that chronic silencing of the prefrontal cortex (PFC) via overexpression of a potassium channel paradoxically increases rsfMRI connectivity between the silenced area and its thalamo‐cortical terminals. Acute chemogenetic silencing of the PFC reproduces analogous patterns of overconnectivity, an effect associated with over‐synchronous fMRI coupling between polymodal thalamic regions and widespread cortical districts. Notably, multielectrode recordings revealed that chemogenetic inactivation of the PFC attenuates gamma activity and increases delta power in the silenced area, resulting in robustly increased delta band coherence between functionallyoverconnected regions. The observation of enhanced rsfMRI coupling between chemogenetically silenced areas challenges prevailing interpretations of functional connectivity as a monotonic index of direct axonal communication, and points at a critical contribution of slow rhythm generators to the establishment of brain‐wide functional coupling.