BackgroundChronic stress exposure increases the risk of mental health problems such as anxiety and depression. The medial prefrontal cortex (mPFC) is a hub for controlling stress responses through communicating with multiple limbic structures, including the basolateral amygdala (BLA) and nucleus accumbens (NAc). However, considering the complex topographical organization of the mPFC neurons in different subregions (dmPFC vs. vmPFC) and across multiple layers (Layer II/III vs. Layer V), the exact effects of chronic stress on these distinct mPFC output neurons remain largely unknown.ResultsWe first characterized the topographical organization of mPFC neurons projecting to BLA and NAc. Then, by using a typical mouse model of chronic restraint stress (CRS), we investigated the effects of chronic stress on the synaptic activity and intrinsic properties of the two mPFC neuronal populations. Our results showed that there was limited collateralization of the BLA- and NAc-projecting pyramidal neurons, regardless of the subregion or layer they were situated in. CRS significantly reduced the inhibitory synaptic transmission onto the BLA-projecting neurons in dmPFC layer V without any effect on the excitatory synaptic transmission, thus leading to a shift of the excitation-inhibition (E-I) balance toward excitation. However, CRS did not affect the E-I balance in NAc-projecting neurons in any subregions or layers of mPFC. Moreover, CRS also preferentially increased the intrinsic excitability of the BLA-projecting neurons in dmPFC layer V. By contrast, it even caused a decreasing tendency in the excitability of NAc-projecting neurons in vmPFC layer II/III.ConclusionOur findings indicate that chronic stress exposure preferentially modulates the activity of the mPFC-BLA circuit in a subregion (dmPFC) and laminar (layer V) -dependent manner.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-023-01050-2.