Project Summary/Abstract Microenvironmental determinants of primary and secondary brain tumor progression are incompletely understood. Neuronal activity is emerging as a critical regulator of brain cancer behavior. We recently showed that active glutamatergic neurons exert a mitogenic effect on high-grade glioma (HGG) through activity- dependent secretion of growth factors - including neuroligin-3 (NLGN3) - which signals to glioma via activation of the PI3K/AKT pathway. Activity-regulated release of NLGN3 promotes growth and is also required for glioma progression, indicating its fundamental role in glioma pathophysiology and is incompletely explained by stimulation of classical oncogenic signaling pathways alone. We previously demonstrated that neuroligin-3 also induces glioma expression of numerous synaptic genes, raising the possibility that gliomas may engage in synaptic communication. Here, we illustrate using electrophysiology and calcium imaging techniques that neuron-glioma interactions include electrochemical communication through bona fide AMPA receptor- dependent neuron-glioma synapses, resulting in excitatory post-synaptic currents and depolarization of glioma cells. This glioma membrane depolarization assessed with in vivo optogenetics promotes glioma progression similarly to the well-established effect in normal neural precursor cells. Concordantly, genetic or pharmacological blockade of glutamatergic signaling inhibits glioma growth. Field recordings demonstrate increased excitability in the glioma-infiltrated brain, emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thereby activity-regulated glioma growth. Together, these findings indicate that synaptic and electrical integration of glioma into glutamatergic neural circuitry promotes tumor progression and elucidates the previously unexplored potential of targeting glioma circuit dynamics for cancer therapy. This proposal aims to further clarify the complex mechanisms of activity-mediated brain tumor growth. Using advanced systems-level neuroscience techniques, the proposed experiments will (1) clarify the role of GABA-ergic signaling in regulating glioma growth through direct neuron-glioma signaling mechanisms and the effect on overall neuronal excitability in the tumor microenvironment; (2) define the dynamic brain-wide circuit mapping of different neuronal subpopulations contributing to glioma progression; (3) expand our understanding of the influence of neuronal activity on non-glial derived secondary brain cancers, thus providing novel insight into the mechanisms metastatic cancers utilize to seed and colonize the brain microenvironment. These studies will develop a framework which maps crosstalk between neurons and brain cancers that may be used to elucidate promising new therapeutic strategies. The experiments and training presented in this proposal will thus answer fundamental questions in this emerging field of the neural regu...