2 2 2 tribromoethanol solution

Under avertin anesthesia (intraperitoneal injection of 2,2,2-tribromoethanol solution; 250 mg/kg body weight; Sigma Aldrich, USA), mice received a stereotaxically guided injection of 40,000 GL261 cells as described (Vannini et al., 2016 (link)(Vannini et al., , 2017)) (link). In C57BL/6J, Thy1-ChR2 and PV-Cre mice, cells were injected either into the primary visual cortex (Vannini et al., 2016) (link) or into the motor cortex (Vannini et al., 2017) (link) in correspondence with the forelimb representation (caudal forelimb area (Alia et al., 2016; (link)Allegra Mascaro et al., 2019) (link)).

One week before the GL261 graft, a cohort of PV-Cre and C57BL/6J mice as controls, received two stereotaxically guided injections of 600 nl of an AAV vector (AAV1.EF1.dflox.hChR2(H134R)-mCherry.WPRE.hGH (Addgene, USA)), one 500 μm anterior and one 500 μm posterior to the site of tumor injection into the motor cortex. PV-Cre mice express Cre recombinase in parvalbuminexpressing neurons. The AAV vector contains the channelrhodopsin-2 (ChR2) gene which is expressed in parvalbumin interneurons through Cre-mediated recombination. We refer to PV-ChR2 to indicate PV-Cre mice injected with the AAV. The two sites of injection were chosen in order to allow the expression of ChR2 in Parvalbumin interneurons located in the motor cortex surrounding the tumor mass. The surgery was performed under avertin anesthesia (intraperitoneal injection of 2,2,2-tribromoethanol solution; 250 mg/kg body weight; Sigma Aldrich, USA).