Glutamate (G-5889; Sigma) stock solution (200 mM) was made in HBS and used at a final concentration of 200 μM. For glutamate perfusions, Alexa Fluor 568 (1-2 μM) was added to the glutamate solution to visualize the start and duration of glutamate exposure. We performed experiments in the presence of TTX (1 μM) to eliminate spiking activity which could influence calcium activity. For calcium imaging experiments we used Fluo-4 NW (F36206; Invitrogen) Calcium Assay Kit. We first incubated cell compartments of the chambers with prewarmed Fluo-4 NW dye mix (0.5×) and 1.25 μM probenecid diluted in HBSS with 20 mM HEPES; the 3 inlet wells for the perfusion channel were filled with HBSS and 20 mM HEPES. The chambers were then incubated at 37°C for >20 min. Before beginning the perfusion, the center inlet well was replaced with 200 μM glutamate diluted in HBSS with 20 mM HEPES.
