The role of early experience to shape neural connectivity has been well demonstrated in sensory systems: during defined time windows called critical periods, neural activity induces structural and synaptic changes that contribute to the functional maturation of brain circuits. These periods of developmental plasticity adapt neural circuits to the environment but are also windows of vulnerability, with long-lasting consequences on behavioral outputs. Growing evidence indicate that associative cortical areas such as the prefrontal cortex (PFC) also show critical periods during early postnatal life: there is substantial evidence that early life stress (ELS) in humans can modify lastingly the function of the PFC and that it increases the risk of developing several psychiatric disorders such as anxiety-related disorders. Exposure to antidepressants during the early postntal period has similar effects and some of the effects of ELS could be due to altered serotonin (5-HT) neurotransmission during development. However, the specific neural circuits and the underlying mechanisms are not known. In this proposal we will take advantage of new tools that are now available to determine whether and how ELS and 5-HT-signaling modify specific PFC neural circuits during early postnatal life. On one hand we have well-characterized mouse models of ELS and of modified 5-HT transmission during postnatal life (PNFLX= postnatal fluoxetine) which both induce functional alterations of the PFC during critical developmental periods. On the other hand, we are able to target specific PFC neural circuits with genetic tools that allow unprecedented selectivity. Finally, the combination of cellular electrophysiological and high-resolution imaging tools allows us to address in a quantitative manner the complex issue of fine-scale neural circuit refinement and evaluate their functional consequences. We focus on a unique PFC neuronal population that is potentially involved in the long-term effects of both ELS and PNFLX mouse models because it expresses the high affinity 5-HT transporter (SERT) during a critical period of maturation of the PFC neural circuits. Unpublished work of P. Gaspar’s group showed that these SERT+ neurons are pyramidal neurons (PNs) that are monosynaptically contacted by GABAergic PV+ (Parvalbumin positive) basket cells (BC) neurons in the PFC (PV-SERT+PN) and that these SERT+PN neurons project subcerebrally to the brainstem, contacting both 5-HT neurons in the raphe nuclei (RN) and dopamine (DA) neurons in the Ventral Tegmental Area (VTA) (SERT+PN-VTA-RN circuit). These neural circuits play important roles in stress and reward processes, thus understanding the factors that influence their functional maturation is particularly relevant to psychiatric disorders. Indeed, both the PV+ PFC interneurons and the monoaminergic neurons (DA, and 5-HT) play a role in vulnerability to psychiatric disorders. We hypothesize that ELS affects the activity-dependent maturation of salient circuits of the PFC, permanently altering its connectivity with RN and VTA, with crucial consequences on adult functionality and anxiety-related behaviors. To tackle this question, we will combine the expertise of Patricia Gaspar on neural circuit development, of Alberto Bacci on cortical microcircuit physiology, and of Camilla Bellone on monoaminergic neuron physiology and development. Three aims will be addressed. 1-Determine whether ELS/PNFLX alters the functional maturation of the PFC microcircuit involving SERT+ PNs and PV cells. 2-Determine whether ELS/PNFLX alters the connectivity of SERT+ PNs with RN/VTA neurons. 3-Investigate the role of changes of PFC neural activity on PFC circuits development and on adult anxiety-related behaviors.