Mni caged glutamate

DIV 8–10 cortical neurons were seeded on plasma-treated, poly-D-lysine-coated Willco dishes, and transfected the day before experiment. Neurons were maintained in Mg²⁺-free ACSF (in mM, 136 NaCl, 2.5 KCl, 2 CaCl₂ 10 D-glucose, 10 HEPES, 2 pyruvate, 1 ascorbic acid, 0.5 myo-inositol) with 10 μM forskolin (Tocris BioSciences), 1 μM TTX (Tocris BioSciences) and, where indicated, 2.5 mM MNI-caged glutamate (Tocris BioSciences, Bristol, UK) for 20′ before uncaging. Following EGFP and Cherry acquisition, 30 pulses (720 nm, 9–13 mW at the objective lens) of 7 ms were delivered at 0.5 Hz at 0.5–1 μm from spine head as in ref. ^{37 (link)}. After 5′, medium was changed to 1 mM MgCl₂ ACSF supplemented with 2% B27 and the same dendrite was imaged after 60′. The mock stimulation was conducted in the same way, except that MNI-glutamate was not added in the medium. For time-course experiments, red and green channels were acquired 20′ and 5′ before the uncaging start. Green channel was acquired at 0.5′, 1′, 2′, 5′, 30′, 60′, and 90′ following uncaging, and red channel was acquired at 5′, 30′, 60′, and 90′. Throughout the whole protocols, neurons were maintained at 37 °C under humidified 5% CO₂ atmosphere. In experiments with translation inhibitors, 5 μM anysomicin (Sigma) was present in the medium all the time staring from the 20′ preincubation.