Acute stress was demonstrated to induce morphological microglial activation in several brain regions including the midbrain periaqueductal gray (PAG), an area that plays important roles in behavioral responses to uncontrollable stress, threat, anxiety, and pain. To determine whether neuronal activation may be involved in the stress-induced microglial activation, the present study investigated the correlation between neuronal activity measured as c-Fos expression and morphological microglial activation in the PAG. Acute stress was followed by morphological activation of microglia and increased c-Fos expression in the PAG but not in the surrounding midbrain. Double immunohistochemistry and topological analysis demonstrated that microglial activation occurred adjacent to responsive neurons. By contrast, lipopolysaccharide (LPS) treatment induced microglial activation even in the absence of neuronal responses in the PGA as well as in the rest of the midbrain. These findings suggest that the mechanism of microglial activation during stress may differ from those of infection or inflammation. It also indicates that the neuronal cells expressing c-Fos protein may play some roles to trigger microglial activation.