Objectives and materials and methods The aims of the present study were (1) to determine the neuronal activation pattern elicited by the group II mGlu antagonist LY341495 and (2) to evaluate the contribution of each group II mGlu subtype by using wild-type (WT) and knockout (KO) mice lacking either mGlu2 or mGlu3. c-Fos expression was used as a marker of neuronal activation. Results and discussion In WT mice, LY341495 induced widespread c-Fos expression in 68 out of 92 brain areas, including limbic areas such as the amygdala, septum, prefrontal cortex, and hippocampus. LY341495-induced cFos response was markedly decreased in the medial part of the central amygdala (CeM) and lateral septum (LS) in mGlu3-KO mice, as well as in the lateral parabrachial nucleus (LPB) in both KO strains. In the majority of investigated areas, LY341495-induced c-Fos expression was similar in KO and WT mice. Analysis of the cellular and subcellular distribution of mGlu2 and mGlu3 revealed a prevailing presence of mGlu3-immunoreactivity in the CeM in glial processes and in postsynapstic neuronal elements, whereas only rare presynaptic axon terminals were found immunoreactive for mGlu2. Conclusion In conclusion, our data indicate that group II mGlu blockade increases neuronal activation in a variety of brain areas, including many stress-and anxiety-related areas. The activation of two key brain areas, the CeM and LS, is mediated via mGlu3, while activation in the LPB involves both subtypes. Moreover, in the majority of investigated areas, LY341495-mediated neuronal activation appears to require a complex cross talk between group II mGlu subtypes or the action of LY341495 on additional receptors.