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The gradient contrast system (GC) we developed gives images of neurons similar to images obtained with differential interference contrast but with superior contrast and light intensity. As in the GC system (Luigs and Neumann), no optical elements for contrast generation had to be placed behind the objective (60×, 0.9 numerical aperture, Olympus); the full power of the UV laser light can reach the neuron. The light of the UV laser was focused through the microscope objective to an optical spot of 1-μm diameter. The power of the laser (Enterprise II, Coherent) was adjusted with its remote control to 5 to 10 mW to evoke a depolarizing response of about 3 to 5 mV at the soma in response to 3-ms shuttered light pulses (Uniblitz shutter; Vincent Associates). Glutamate was applied by photolysis (wavelength = 351 to 364 nm) of γ-(α-carboxy-2-nitrobenzyl) (CNB)-caged glutamate (Molecular Probes) added at a concentration of 0.25 to 0.5 mM to the superfusion solution, which was oxygenated in a recirculation system. No indications for effects of spontaneously hydrolyzed γ-CNB-caged glutamate were found. All photostimulation experiments were started at least 15 min after the addition of caged glutamate and TTX.
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3, and 25 mM glucose (pH 7.38; 20° to 22°C). Whole-cell recordings from visually identified somata of layer V pyramidal neurons were made with a standard intracellular amplifier (npi) in bridge mode. Patch-clamp electrodes with open-tip resistances of 4 to 7 megaohms were used that contained the following: 130 mM K-gluconate, 5 mM KCl, 0.5 mM EGTA, 2 mM Mg-adenosine triphosphate, 10 mM Hepes, and 5 mM glucose (pH 7.2) (21). Data were stored and analyzed with a Macintosh-based recording system and standard software (Pulse, HEKA).
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We thank K. Lee for comments on this manuscript. Supported by a grant from the Sonderforschungsbereich 391.
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