We demonstrate a novel role of phospholipase D in M1-dependent rodent cortical plasticity and M1 PAMs that do not couple to phospholipase D have functionally distinct effects on cortical plasticity than non-biased M1 PAMs. AbstractHighly selective positive allosteric modulators (PAMs) of the M1 subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach for the potential improvement of cognitive function in patients suffering from Alzheimer's disease and schizophrenia. M1 PAM discovery programs have produced a structurally diverse range of M1 PAMs with distinct pharmacological properties, including different levels of agonist activity and differences in signal bias. This includes the recent discovery of novel biased M1 PAMs that can potentiate coupling of M1 to activation of phospholipase C but not phospholipase D (PLD). However, little is known about the role of PLD in M1 signaling in native systems and it is not clear whether biased M1 PAMs will display differences in modulating M1-mediated responses in native tissue. We now report a series of studies using novel PLD inhibitors and PLD knockout mice to show that PLD is necessary for the induction of M1-dependent long-term depression (LTD) in the prefrontal cortex (PFC).Importantly, biased M1 PAMs that do not couple to PLD not only fail to potentiate orthosteric agonist-induced LTD but also block M1-dependent LTD in the PFC. In contrast, biased and nonbiased M1 PAMs act similarly in potentiating M1-dependent electrophysiological responses that are PLD-independent. These findings demonstrate that PLD plays a critical role in the ability of M1 PAMs to modulate certain CNS functions and that biased M1 PAMs function differently in brain regions implicated in cognition.