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Background Glioblastoma (GBM) is the most common primary brain tumor, with a poor prognosis of 14-17 months median survival. The standard of care, consisting of surgery, chemotherapy, and radiation has not changed in over 20 years, and no phase III immunotherapy clinical trial has been successful. Recent studies determined that in GBM, tumor cells integrate into neural networks, forming synapses with neurons, enhancing neuronal firing and facilitating tumor growth. However, the impact of neuronal activity on the tumor microenvironment remains poorly understood and may have therapeutic importance. We sought to understand if neuronal activity alters the function of immune cells in GBM using both murine models and freshly acquired human GBM tissue from surgical specimens.Methods and Results We first used chemogenetic stimulation (AAV5-SYN1-hM3Dq) of an RCAS-tva mouse glioblastoma model (Ntv-a Ink4a-Arf-/-) to identify which tumor immune populations are impacted by enhanced neuronal firing in vivo. Using high-dimensional spectral flow cytometry, we found an increase in microglia percentages following stimulation. We then used human GBM tissue to study the putative neuron-myeloid interaction. Immunofluorescent staining of these tumors showed that approximately 50% of the Iba1+ myeloid cells co-localized with the presynaptic neuronal marker synapsin 1, confirming the neuron-myeloid interaction. We then sorted CD11b+ myeloid cells from these patient samples. Bulk RNA-seq revealed high expression of neurotransmitter receptors, predominantly the G i protein-coupled P2Y12 receptor for ADP. Calcium fluorescence imaging and calcium flow cytometry showed that the cells were primarily responsive to ADP among the neurotransmitters examined, including glutamate, GABA, norepinephrine, acetylcholine, and dopamine. Furthermore, the phagocytic ability of these cells improved with the addition of ADP. Analysis of bulk RNA-seq from 286 brain tumor patients found that P2Y12 had a clear positive correlation with M1-like markers and a negative correlation with M2-like markers greater than all other neurotransmitter receptor genes and others such as ADRB2, CX3CR1, TMEM119, TREM2, and CSF1R. To understand how the ADP-P2Y12 axis affects myeloid phenotypic function in vivo, we conducted flow cytometry on our SB28 mouse brain tumor tissues. Based on functional marker expression, we found that, compared to P2Y12-negative microglia, P2Y12-positive microglia had an increased M1-like and decreased M2-like phenotype.Conclusions Our findings strongly suggest that neuronal excitation impacts microglia in the GBM microenvironment, primarily through an ADP-P2Y 12 axis. Furthermore, our data suggests that P2Y12 may play a role in promoting an M1-like phenotype in microglia and could be an important target for enhancing response to immunotherapy.Ethics Approval All human tissue samples and experiments were conducted according to the approved protocols by the University of Cambridge under REC reference 23/EE/0241 and IRAS project ID 335210. Each participant was given informed consent prior to taking part in any research studies. All animal work was performed according to the National Cancer Institute-Bethesda Animal Care and Use Committee’s protocols NOB-024 and NOB-026.