Paradoxical widespread c-Fos expression induced by a GABA agonist in the forebrain of transgenic mice with ectopic expression of the GABAA α6 subunit

• Transgenic Thy1α6 mice overexpress extrasynaptic GABAA receptors in the forebrain.
• Thy1α6 mice show enhanced sensitivity to anxiolytic effects of gaboxadol.
• Gaboxadol paradoxically increased c-Fos expression in the Thy1α6 forebrain.
• Especially the central extended amygdala and septum displayed enhanced activation.
• Thus, activation of neurons in these regions appeared to mediate the gaboxadol-induced anxiolysis.

A GABA-site agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) at 3 mg/kg induces strong anxiolytic response in a transgenic Thy1α6 mouse line ectopically expressing the GABAA receptor α6 subunit gene under the Thy-1.2 promoter. Now, we compared brain activation patterns between Thy1α6 and wild-type mice to identify brain structures potentially mediating this anxiolytic response. Acutely efficient anxiolytics such as benzodiazepines typically depress most brain regions while activating specifically neurons within the central extended amygdala. Gaboxadol treatment (3 mg/kg, i.p., 2 h) induced a significant increase in c-Fos expression selectively in many Thy1α6 brain regions including the limbic cortex, anterior olfactory nucleus, septal area and central and basolateral nuclei of amygdala. It failed to activate the lateral part of mediodorsal thalamic nucleus (MDL) in the Thy1α6 mice that was activated in the wild-type mice. Detailed mapping of the α6 subunit mRNA by in situ hybridization revealed expression in the middle layers of the isocortex, olfactory areas, hippocampal formation and basolateral nucleus of amygdala (BLA) in the Thy1α6 forebrain. The ligand autoradiographies (t-butylbicyclophosphoro[35S]thionate ([35S]TBPS) and [3H]Ro 15-4513) revealed high levels of pharmacologically active extrasynaptic α6β and α6βγ2 GABAA receptors in these same areas. However, c-Fos induction by gaboxadol treatment in Thy1α6 brain was not restricted to areas highly expressing the α6-containing GABAA receptors suggesting that indirect pathways lead to the paradoxically widespread activation. Interestingly, the activation pattern by gaboxadol at the dose that is anxiolytic in Thy1α6 mice resembled closely that observed after various fear- and stress-provoking challenges. However, our results are consistent with a recent observation that optogenetic activation of specific neuronal pathways in the extended amygdala mediates anxiolytic responses. Our results suggest that the widespread neuronal inhibition as typically associated with benzodiazepines is not the exclusive mechanism of anxiolysis.