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. 2007 Jun 20;27(25):6601-4.
doi: 10.1523/JNEUROSCI.1519-07.2007.

4-Carboxymethoxy-5,7-dinitroindolinyl-Glu: an improved caged glutamate for expeditious ultraviolet and two-photon photolysis in brain slices

Graham C R Ellis-Davies  1 , Masanori Matsuzaki, Martin Paukert, Haruo Kasai, Dwight E Bergles
Affiliations

4-Carboxymethoxy-5,7-dinitroindolinyl-Glu: an improved caged glutamate for expeditious ultraviolet and two-photon photolysis in brain slices

Graham C R Ellis-Davies et al. J Neurosci. .
No abstract available

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Figures

Figure 1.
Figure 1.
Synthesis of CDNI-Glu. The reagents and conditions are listed as follows: (a) ethylbromoacetate, K2CO3, acetone, room temperature (RT = 23-25°C), 18 h; (b) NaBH3CN, AcOH; (c) as Papageorgiou et al., 2004; (d) dicyclohexylcarbodiimide (R = ethyl) or 1-(3-dimethylaminopropyl)-3-ethycarbodimide (R = methyl); (e) claycop, acetic anhydride, CCl4, RT; (f) HNO3, acetic anhydride, RT; (g) LiOH, THF, RT; (h) TFA, RT; (i) NaOH, MeOH, RT; (j) NaNO3 (1.2 equ), TFA, RT, then HPLC isolation of 5-isomer; (k) NaNO3 (20 equ), TFA, RT. Full details of the synthesis are in the supplemental material (available at www.jneurosci.org). The inset box shows NMRs in D2O of the amino acid portion of CDNI-Glu before and after photolysis: the caging chromophore is photolyzed (peak marked with * disappears), and glutamate is cleaning released.
Figure 2.
Figure 2.
Comparative uncaging of CDNI-Glu and MNI-Glu on pyramidal neurons in acutely isolated cortical brain slices using UV irradiation. A, The caged glutamates (concentration of 0.2 mm) were locally perfused onto a pyramidal neuron through a wide bore application pipette. The photolysis beam (333.6–363.8 nm; diameter, 50 μm; 30 mJ/cm2) was focused through a 40× water objective lens (for full details, see Huang et al., 2005). B, Superimposed average traces comparing responses to photolysis of CDNI-Glu (in red) and MNI-Glu (in black) from five cells (top traces). The traces taken from the recording in A are highlighted by the arrow. Normalization of the response to MNI-Glu to the peak amplitude of the response to CDNI-Glu uncaging (bottom traces) is shown. C–F, Comparison of the peak currents, total charge, rise times, and decay times of the evoked currents from uncaging CNDI-Glu and MNI-Glu (the lines in E and F connect responses collected from individual cells). p values were determined using the Student's paired t test.
Figure 3.
Figure 3.
Comparative uncaging of CDNI-Glu and MNI-Glu on acutely isolated hippocampal brain slices using two-photon excitation. A, The caged glutamates ([CDNI-Glu] = 3.5 mm; [MNI-Glu] = 10 mm) were locally perfused onto the same hippocampal CA1 neuron through an application pipette. A small dendritic region (Ag) was selected for two-photon mapping according to our standard procedure (Matsuzaki et al., 2001). The region of interest was divided into 13 × 16 pixels (size 0.71 μm). The photolysis beam (mode-locked Ti:sapphire laser tuned to 720 nm) was focused through a 60× water objective lens (NA 0.9). Uncaging of MNI-Glu (Aa–c) and CDNI-Glu (Ad–f) at three different energies (a, d, 3.9 mW for 1.2 ms; b, e, 3.9 mW for 0.8 ms; c, f, 2.7 mW for 0.8 ms) produced different mapping intensities. Currents are displayed according to the pseudo-color scale. B, Quantitative comparison of the photoevoked currents in A with power (P = 3.9 or 2.7 mW), shutter time (T = 1.2 or 0.8 ms), and cage concentration (C) showed that CDNI-Glu (blue line) was 4.2 times more photosensitive than MNI-Glu (black line). C, Repetitive two-photon uncaging of CNDI-Glu (7 mm) or MNI-Glu (12 mm) at single spine heads using a short shutter open time (10 μs) and high laser intensities [CNDI-Glu, 22 mW (blue) and MNI-Glu, 48 mW (black)]. Energy per flash for CDNI-Glu was approximately the same as in Af (n = 9; ±SEM). D, Three examples of current traces induced by photolysis of CNDI-Glu (from C). Black, blue, and green traces correspond to the first, 10th and 20th trials, respectively. The top trace showed the time of the illumination.
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