Image acquisition and two-photon glutamate uncaging

A custom-built two-photon microscope with two Ti: sapphire lasers (Spectra-physics) was used. One laser was tuned at a wavelength of 920 nm to excite eGFP. All samples were imaged using <2 mW laser power measured at the objective (60×, 0.9 numerical aperture; Olympus). The second laser was tuned to 720 nm to uncage 4-methoxy-7-nitroindolinyl-caged-l-glutamate (MNI-caged glutamate) with a train of 6 ms, 5 mW pulses (30 times at 0.5 Hz) near the head of the spine of interest. The beams were combined and passed through the same set of scan mirrors and objective. Fluorescence signal from a cooled PMT (Hamamatsu) was acquired using a data acquisition board controlled by Scanimage software. Two-photon glutamate uncaging was performed at 24–26°C (room temperature) in Mg²⁺-free artificial CSF (ACSF; 127 mm NaCl, 2.5 mm KCl, 4 mm CaCl₂, 25 mm NaHCO₃, 1.25 mm NaH₂PO₄, and 25 mm glucose) containing 1 μm tetrodotoxin (TTX) and 4 mm MNI-caged glutamate aerated with 95% O₂ and 5% CO_2. We imaged one to five spines/neuron and at least two neurons/animal.