Distinct activity in prefrontal projections promotes temporal control of action

Prefrontal neurons exhibit diverse activity during cognitive functions such as working memory, attention, and timing; however, the importance of this heterogeneity is unclear. Our goal was to better understand the diversity of prefrontal activity through anatomical connectivity. We harnessed circuit-specific tools in mice to capture activity within prefrontal projections during interval timing, a highly translational cognitive process that requires working memory for temporal rules and attention to the passage of time to estimate a temporal interval of several seconds. We used neuronal recordings to capture prefrontal activity during interval timing, with major patterns characterized by monotonic time-dependent ramping over a temporal interval. We then leveraged retrograde viruses to interrogate prefrontal cortex (PFC) projections to the mediodorsal thalamus (PFC-MD) and the dorsomedial striatum (PFC-DMS). We report three main findings. First, circuit-specific fiber photometry revealed that PFC-MD and PFC-DMS activity encoded distinct temporal signals, with PFC-MD projections ramping down and PFC-DMS ramping up to interval timing response times. Second, circuit-specific inactivation revealed that suppressing PFC-DMS projections disrupted animals' internal estimates of time. Third, circuit-specific single-nucleus RNA sequencing of projection-defined prefrontal neurons revealed distinct transcriptomic profiles of PFC-MD and PFC-DMS projections, with enrichment of cortical layer-associated genes as well as genes such as Cux2, Camk2n1, Htr4, and Foxp2. These data suggest that differences in gene expression and connectivity distinguish prefrontal activity during interval timing. These findings advance our fundamental understanding of prefrontal function and dysfunction in human disease.