Glioblastoma is a highly malignant tumor with poor prognosis. Intercellular signaling has been proposed to be directly involved in glioblastoma proliferation, motility and invasion. In particular, (i) glioblastoma growth and infiltration are associated to deregulated astrocytes-neuron signaling; (ii) glioblastoma induces astrocytes function alterations leading to modification of the inter and intra cellular communication and neuronal hyperactivity and epileptic seizures; (iii) glutamate release in peri-tumoral region excite glutamate receptors on glioma cells promoting tumor growth and invasiveness [1-3]. Connexins (Cx), are transmembrane proteins essential for cell-to-cell communication, that have been linked to glioma cell migration and invasion, since they can mediate the release of signaling molecules, such as ATP [2]. A body of evidence indicates that Cx43 hemichannels play a role in hyperexcitability and epileptogenesis, early pathophysiological hallmark of malignant glioma [4], via deregulated Ca2+-dependent glutamatergic signaling [5]. With this study we aim to improve our understanding of neuron-astrocyte network dysregulation in a glioblastoma mouse model in presence and absence of astrocytic Cx43, by evaluating in vivo the intercellular signaling mediated by Ca2+ and glutamate within cancer cells and in neuron-astrocyte networks of the peri-tumoral region by means of multicolor two photon excitation microscopy imaging through a glass cranial window for longitudinal monitoring.1. Venkataramani, V., et al., Cell, 2022.2. De Vuyst, E., et al., Cell Calcium, 2009.3. Walrave, L., et al., Glia, 2018.4. Huberfeld, G. and C.J. Vecht, Nat Rev Neurol, 2016.5. Lange, F., J. Hornschemeyer, and T. Kirschstein, Cells, 2021.