Beyond the Relay: Mediodorsal Thalamic Regulation of Prefrontal Cortex in Cue Detection

Abstract

The mediodorsal thalamus (MD) is a critical node within thalamocortical networks supporting prefrontal-dependent cognition, yet whether its projections to distinct prefrontal subregions constitute functionally specialized pathways has not been directly examined. Here we combined retrograde anatomical circuit mapping with projection-specific fiber photometry to characterize the structural and functional organization of MD connectivity with the prelimbic cortex (PRL), anterior cingulate cortex (ACC), and orbitofrontal cortex (OFC) in mice. Retrograde tracing revealed that MD neurons projecting to PRL and ACC originate from anatomically segregated subpopulations within the lateral MD subdivision (MDL), organized along its anterior-posterior axis and forming distinct reciprocal loops with each cortical target. Projection-specific calcium imaging during Pavlovian cue-reward conditioning revealed divergent dynamics across pathways: MD-ACC terminals underwent pronounced learning-dependent temporal sharpening, with response precision increasing across acquisition and trial-by-trial dynamics becoming coupled to behavioral performance, while MD-PRL terminals showed stable, sustained cue-evoked responses that did not reshape across learning. MD-OFC terminals showed no learning-related changes during Pavlovian conditioning, consistent with OFC's established role in action-outcome rather than cue-association learning, motivating a targeted examination of MD-OFC dynamics during an instrumental task, in which the animal must explicitly use the cue to guide behavior. During instrumental performance, MD-OFC terminals exhibited robust trial-phase-specific modulation that was absent during Pavlovian conditioning, including history-dependent pre-trial baseline signals that predicted upcoming behavioral performance, post-outcome signals encoding reward identity, and across-trial carryover effects linking outcome experience to future circuit state. Together, these findings demonstrate that MD is a functionally heterogeneous nucleus whose anatomically segregated projections support complementary aspects of cue-guided learning and flexible behavior, positioning MD as an active regulator of prefrontal computation rather than a passive thalamocortical relay.