Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C079

Oral Communications

Astrocytic glutamate clearance is regulated by neuron-dependent Notch signalling as well as cAMP

A. C. Todd1,2, P. Hasel1,2, P. S. Baxter1,2, D. J. Wyllie1, G. E. Hardingham2,1

1. Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, Midlothian, United Kingdom. 2. UK Dementia Research Institute at Edinburgh, University of Edinburgh, Edinburgh, United Kingdom.


Glutamate clearance is an important function of astrocytes, preventing excitotoxic neurotransmitter build-up. The two astrocytic transporters responsible for this glutamate clearance are EAAT1 (Slc1a3) and EAAT2 (Slc1a2). Neurons have been 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, such as Alzheimer's disease, elucidating the mechanisms that regulate these transporters is of clinical importance1,2. Using a novel mixed-species RNAseq approach, our lab has recently shown that numerous astrocytic genes are regulated by neurons, including glutamate transporter and Notch pathway genes3. We used these findings to investigate astrocytic EAAT function and the mechanism behind their regulation. Primary cultures of cortical mouse astrocytes (from E19.5 pups) were grown either alone as monoculture, or cocultured with cortical rat neurons (from E21.5 pups)3. 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 currents 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 signalling. 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 (ctrl)). 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 increase EAAT function, monoculture astrocytes were treated with 8-Br-cAMP (0.5 mM). After 1wk of treatment there was an increase in astrocytic EAAT currents (I = 59±12 vs 13±4 pA, p<0.01, n=25 (8-Br), n=20 (ctrl)). This enhancing effect was also observed in coculture astrocytes, and separately in astrocytes treated with a combination of forskolin and IBMX to boost cellular cAMP levels. 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.

Where applicable, experiments conform with Society ethical requirements