The vulnerability to stress and mood disorders is thought to have a developmental origin. Converging evidence indicates that prefrontal cortex (PFC) circuits engaged in cortico-limbic top-down control are key in the developmental etiology of mood disorders. The neural circuit connecting the PFC to the dorsal raphe nucleus (DRN) is critically involved in stress-coping responses and mood control, and represents the main source of brain serotonin (5-HT). During mouse development there is a critical period [postnatal days (P) 2 to 14] when environmental factors can influence neurodevelopmental trajectories with long-lasting consequences for adult life. The early-life stress of maternal separation (MS) is a validated model that causes adult emotional alterations. We investigate how the early PFC-to-DRN circuit is formed and refined, and how dysregulation of its neurodevelopment is affected in the MS model. We evaluated alterations in the synaptic connectivity of the PFC-to-DRN circuit using the high-resolution microscopy technique Array Tomography and the activation of DRN 5-HT neurons was assessed by cFos immunostaining. To investigate possible physiological correlates accompanying morphological changes we performed ex-vivo patch clamp recordings on both 5-HT and GABA DRN neurons of MS mice at these different developmental ages. Our work indicates that maternally-separated mice have alterations in the PFC-to-DRN circuit and 5HT neuron stress-dependent activation.