Two-Photon Imaging and Glutamate Uncaging

Simultaneous 2-photon imaging and uncaging was performed using a dual galvanometer-based scanning system (Prairie Technologies, Middleton, WI) using two Ti:sapphire pulsed lasers (MaiTai, Spectra-Physics, Santa Clara, CA), one tuned to 840 nm for imaging cell morphology, and another tuned to 720 nm for photolysis of MNI-caged-L-glutamate. Neurons were visualized using an Olympus BX51WI objective (60x, 0.9 NA; Olympus, Melville, NY). Two-photon glutamate uncaging was carried out based on previously published methods (Gasparini and Magee, 2006 (link); Losonczy and Magee, 2006 (link); Branco and Häusser, 2011 (link)). MNI-caged-L-glutamate (12 mM, Tocris Cookson, UK) was dissolved in (in mM): NaCl 125, KCl 2.5, HEPES 10, CaCl ${}_2$ 2, MgCl ${}_2$ 1, glucose 25, and puffed locally. To block NMDA receptors (Figure 5E,J, blue squares), 500 μM D-AP5 was included in the glutamate puffing pipette.