Mni caged l glutamate

The chemical reagents used for this study were MNI-caged-L-glutamate (Tocris). Stock solutions of 50 mM were stored at −20°C with concentrations of 1% DMSO. Working solutions were diluted to 10 μM. All embryos were dechorionated at 24 hpf and incubated with 3 mL egg water until desired treatment time [35 (link)]. Fish were treated at 4 dpf with 10 μM caged glutamate 1 hour before imaging. Control fish were incubated with 1% DMSO in egg water 1 hour before imaging.

All drugs were purchased from Sigma Aldrich (St. Louis, MO), or Tocris (Bristol, UK). SKF 81297 hydrobromide (Tocris, 1447) was applied either as puff (500 µM) or flow in (10 µM) depending on experiment type. All other drugs were bath applied: SCH-39166 hydrobromide (Tocris,2299), SCH-23390 hydrochloride (Tocris, 0925), SKF-83566 hydrobromide (Tocris, 1586), Strychnine (Sigma-Aldrich, 8753), Tetraethylammonium chloride (Sigma-Aldrich, 86614), TTX (Tocris Bioscience, 1069), (RS)-CPP (Tocris Bioscience, 0173), NBQX (Tocris Bioscience, 0373), SR 95531 hydrobromide (Tocris Bioscience, 1262), MNI-caged-L-glutamate (Tocris Biosciences 1490), L-741,626 (Tocris Bioscience, 1003), Pyr-3 (Tocris,3753), prazosin (Sigma-Aldrich, P7791), propranolol (Sigma-Aldrich, 40543), and quinpirole hydrochloride (Tocris Bioscience, 1061). For experiments requiring pharmacological agents dissolved in DMSO the concentration never exceeded 0.02% DMSO.

Concentrated stock solutions of various pharmacological agents were initially prepared and diluted in physiological saline to a final concentration before use. For uncaging experiments, MNI-caged-L-glutamate (Tocris, Ellisville, MO) or and MNI-L-glutamate trifluoro acetate (Femtonics, Hungary) were prepared each day at final concentration in physiological solution. All agonists and antagonists were purchased from Sigma (St. Louis, MO) or Tocris (Ellisville, MO). The presence or absence of tetrodotoxin (TTX) is provided for each experiment.

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.

4-Aminopyridine, PTX, poly-L-lysine, D(−)-2-amino-5-phosphonopentanoic acid, Trolox, DAPI nuclear dye, Nocodazole, and Ciliobrevin D were purchased from Sigma. Bicuculline methiodide, MNI-caged-L-glutamate, Anisomycin were purchased from Tocris Bioscience. Leptomycin B (10 nM) was purchased from Beyotime. Peptides used are Tat-APPL1₁₃ (YGRKKRRQRRRRRASEKQKEIERVKEK) and Tat-APPL1_Scr (YGRKKRRQRRRRRASEKQKEIEAAAAA). The following antibodies were used: anti-APPL1 (sc-67402) and anti-Rab5 (sc-46692) from Santa Cruz Biotechnology, anti-pERK (4370S) and anti-GAPDH (2118) from Cell Signaling Techonology, anti-HDAC2 (ab32117), anti-Importin α1 (ab84440), anti-Histone H4K5 (ab51997), anti-Histone H4K12 (ab177793), anti-APPL2 (ab95196), and anti-Histone H4 (ab10158) from Abcam, anti-MAP2 (M9942, M3696), anti-FLAG (F1804), and anti-APPL1 (1409089) from Sigma–Aldrich, and anti-pCREB (06-519 and 04-218) from Millipore. Glutathione sepharose beads and protein A sepharose beads were purchased from GE Healthcare. Phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma. Horseradish peroxidase (HRP)-linked goat anti-mouse immunoglobulin G (IgG), goat anti-rabbit IgG, and donkey anti-goat IgG, secondary antibodies conjugated to Dylight (488 or 555), and chemiluminescence kit were purchased from Pierce.