Uncaging of caged glutamate

Single-photon laser photolysis of 4-methoxy-7-nitroindolinyl-caged glutamate (MNI-Glu) was performed using a 405 -nm continuous-wave (CW) laser³⁹. An uncaging laser (OBIS 405LX-100, Coherent) was combined with an imaging line of a Nikon Structured Illumination Microscope system (N-SIM) using a dichroic mirror. The uncaging laser was controlled separately using the Coherent Connection software (Coherent) via transistor–transistor logic (TTL) generated by an Arduino UNO microcontroller. The uncaging laser was aligned to the center of the imaging field before each experiment.

For glutamate uncaging, hippocampal neurons were maintained at 37 °C in Mg²⁺-free Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 4 mM CaCl₂, 0 mM MgCl₂, 25 mM HEPES, and 30 mM glucose; pH 7.4) with 1 μM tetrodotoxin (TTX; WAKO) and 500 μM MNI-Glu (Tocris). Medium-size spines with clear heads and necks were selected for induction of structural change. Single-photon glutamate uncaging was performed by 2 msec pulses repeated at 1 Hz for 1 min with the center of the focused laser beam 1–2 μm away from the tip of the spine³⁹. Precise control of the sample position was achieved by operating a motorized XY stage with N-SIM encoders. Laser intensity was set to 0.05–0.15 mW at the back aperture of the objective lens.