Glutamate clearance is an important function of astrocytes, and is essential for preventing excitotoxic neurotransmitter build-up. The two astrocytic transporters responsible for glutamate clearance are EAAT1 (Slc1a3) and EAAT2 (Slc1a2). Neurons have been previously shown to induce astrocytic expression of Slc1a2 and Slc1a3 genes; however, the pathways mediating this induction are unclear. As altered astrocytic glutamate uptake is implicated in numerous neurodevelopmental and neurodegenerative diseases, elucidating the mechanisms that regulate these transporters is of clinical importance. Using a novel mixed-species RNA sequencing approach, our lab has recently shown that numerous astrocytic genes are regulated by neurons, including glutamate transporter and Notch pathway genes (Hasel et al., 2017). We used these findings to investigate astrocytic EAAT function and the mechanism behind their regulation. Primary cultures of cortical mouse astrocytes (prepared from E19.5 pups) were grown either alone as monoculture, or cocultured with cortical rat neurons (prepared from E21.5 pups), as detailed in Hasel et al., 2017. Glutamate transporter function was measured electrophysiologically in astrocytes by transporter currents induced by application of the EAAT agonist L-aspartate (200 μM) in the presence of AP-5 (100 μM). Current identity was confirmed by the EAAT inhibitor TFB-TBOA (20 µM). Results are given as mean ± S.E.M. assessed by unpaired t-test. There was a significant increase in EAAT current in cocultured compared to monocultured astrocytes (I = 3.2±0.9 vs 34.2±5.4 pA, p<0.01 n=19 (mono), n=12 (co)). To investigate the involvement of Notch signalling in this induction, the γ-secretase inhibitor DAPT was applied to cocultured astrocytes to inhibit Notch pathway activation. DAPT treated astrocytes had significantly lower EAAT currents compared to untreated cells (I = 9.9±1.8 vs 34.2±5.4 pA, p<0.01, n=10 (DAPT), n=12 (control)). To confirm the role of Notch signalling, monocultured astrocytes were transfected with the constitutively active Notch effector CBF1-VP16 or a globin control. Astrocytes expressing CBF1-VP16 had greater EAAT currents than controls (I = 15.8±2.8 vs 4.2±1.2 pA, p<0.01, n=10 (CBF1) n=8 (globin)). Finally, cyclic AMP (cAMP) analogues have been shown to increase astrocytic EAAT expression. To determine the ability of cAMP to likewise increase EAAT function, monoculture astrocytes were treated with 8-Br-cAMP. After 1wk of treatment there was a significant increase in astrocytic EAAT currents compared to control (I = 144±18 vs 23±9 pA, p<0.01, n=7 (8-Br), n=6 (control)). These data show that both neuron-dependent Notch signalling and cAMP can regulate astrocytic EAAT function, offering new clinically relevant targets to explore to alter astrocytic glutamate uptake capacity.