Patients suffering from major depression have repeatedly been reported to have dysregulations in hypothalamus–pituitary–adrenal (HPA) axis activity along with deficits in cognitive processes related to hippocampal and prefrontal cortex (PFC) malfunction. Here, we utilized three mouse lines selectively bred for high (HR), intermediate, or low (LR) stress reactivity, determined by the corticosterone response to a psychological stressor, probing the behavioral and functional consequences of increased vs. decreased HPA axis reactivity on the hippocampus and PFC. We assessed performance in hippocampus- and PFC-dependent tasks and determined the volume, basal activity, and neuronal integrity of the hippocampus and PFC using in vivo manganese-enhanced magnetic resonance imaging and proton magnetic resonance spectroscopy. The hippocampal proteomes of HR and LR mice were also compared using two-dimensional gel electrophoresis and mass spectrometry. HR mice were found to have deficits in the performance of hippocampus- and PFC-dependent tests and showed decreased N-acetylaspartate levels in the right dorsal hippocampus and PFC. In addition, the basal activity of the hippocampus, as assessed by manganese-enhanced magnetic resonance imaging, was reduced in HR mice. The three mouse lines, however, did not differ in hippocampal volume. Proteomic analysis identified several proteins that were differentially expressed in HR and LR mice. In accordance with the notion that N-acetylaspartate levels, in part, reflect dysfunctional mitochondrial metabolism, these proteins were found to be involved in energy metabolism pathways. Thus, our results provide further support for the involvement of a dysregulated HPA axis and mitochondrial dysfunction in the etiology and pathophysiology of affective disorders.