Abstract

The medial prefrontal cortex (mPFC) and the dorsomedial striatum (dmStr) are linked to working memory (WM) but how striatum-projecting mPFC neurons contribute to WM encoding, maintenance, or retrieval remains unclear. Here, we probed mPFC[->]dmStr pathway function in freely-moving mice during a T-maze alternation test of spatial WM. Fiber photometry of GCaMP6m-labeled mPFC[->]dmStr projection neurons revealed strongest activity during the delay period that requires WM maintenance. Demonstrating causality, optogenetic inhibition of mPFC[->]dmStr neurons only during the delay period impaired performance. Conversely, enhancing mPFC[->]dmStr pathway activity--via pharmacological suppression of HCN1 or by optogenetic activation during the delay-- alleviated WM impairment induced by NMDA receptor blockade. Consistently, cellular-resolution miniscope imaging resolved preferred activation of >50% mPFC[->]dmStr neurons during WM maintenance. This subpopulation was distinct from neurons showing preference for encoding and retrieval. In all periods, including the delay, neuronal sequences were evident. Striatum-projecting mPFC neurons thus critically contribute to spatial WM maintenance.