4.7. Electrophysiology and Optogenetics

Electrophysiological recordings were made from DIV8-DIV12 hippocampal neurons transfected with light-gated channels/pumps on DIV3. Cultures were transferred to a custom-made imaging chamber, adapted to fit with the motorized stage, in extracellular solution (in mM: 148 NaCl, 2.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 7 glucose in dH2O; 300 mOsm, pH 7.4). To isolate ChR2 photocurrents and block spontaneous network activity, 1μM TTX (Alomone Labs; cat. no. T–550), 50μm APV (Abcam; cat. no. ab120003), 10μM CNQX (Sigma; cat. no. C239), and 10μm gabazine (Abcam; cat. no. ab120042) were added to the extracellular solution. Patch pipettes with 3 to 5MΩ resistance were made from fire polished borosilicate capillaries (Harvard Apparatus; cat. no. 300060, OD1.5mm×ID0.86mm) with a P–97 Micropipette Puller (Sutter Instruments). Silver wire electrodes were chlorinated with a 2 M KCl solution using an ACl-01 apparatus (npi electronic). Patch pipettes were backfilled with 7-μl internal solution (in mM: 130 potassium gluconate, 8 KCl, 2 CaCl2, 1 MgCl2, 10 EGTA, 10 HEPES, 2 Mg-ATP, 0.3 GTP-Na; 290 to 295 mOsm, pH 7.3), using 20μl microloader pipette tips (eppendorf; cat. no. 5242 956.003). A pipette holder was controlled using a MPC-385-2 micromanipulator system (Sutter Instruments). Whole-cell patch-clamp recordings were obtained with an EPC10 USB double patch-clamp amplifier and Patchmaster software (HEKA) at 25 kHz sampling intervals.

All optogenetic experiments were conducted on an inverted Zeiss Axio Observer.Z1 microscope equipped with a 405-nm 50-mW diode laser, 488-nm 100-mW OPSL, 561-nm 40-mW diode laser, and 639-nm 30-mW diode laser, 20× LD A-Plan, 40× and 63× EC Plan-NEOFLUAR and 100× α Plan-APOCHROMAT objectives, a TIRF slider module, a laser manipulation DirectFRAP module, and an Evolve 512 EMCCD camera (Photometrics). An additional 594-nm 100-mW DPSS laser (Rapp OptoElectronic GmbH; Hamburg, Germany) was added to the epifluorescence light path with a 940-μm light guide, together with custom photomasks, complementing the existing DirectFRAP photomasks by adding a 594-nm border (2μm for 100× objective) around the DirectFRAP photomasks. A photomask of a 150-μm wide area or no photomask was used for initial single wavelength full-field illumination tests. All lasers were operated by TTL pulses delivered by the HEKA amplifier.

Laser powers were determined with a laser power meter (Fieldmate) and a silicon OP-2 VIS optical sensor (Coherent GmbH; Dieburg, Germany) at the back aperture of the 40× and 100× objectives, using two aluminum masks to represent the respective exit pupil diameter, 10.7 and 4.8 mm. There was no difference in the measured power between those two masks. Laser intensities were measured after 4 to 6 h of use and with the TIRF/FRAP beamsplitter set at 50% TIRF/50% FRAP. The measured laser powers were subsequently corrected for the respective transmission of the objective. Laser output generally fluctuated ±30μW and the measurement error was previously determined to be ∼10%. 594 nm laser intensity was measured using the 200-μm light guide and respective powers for the 100-μm light guide were extrapolated. For initial tests of light-gated channel candidates, we used wide-field illumination with the 40× objective at power densities of 405 nm 87mW/cm2, 488 nm 296mW/cm2, 561 nm 146mW/cm2, and 594 nm 801mW/cm2 or 1.08W/cm2. In experiments in which the light path was adjusted to increase 594-nm laser power (by exchanging the 940-μm light guide for a 100-μm light guide, and the DirectFRAP beamsplitter for an AHF beam splitter), power densities using the 40× objective were 405 nm 27 to 65mW/cm2, 488 nm 228mW/cm2, 561 nm 133mW/cm2, and 594 nm 129.75W/cm2 or 267W/cm2. Because the 594-nm photomasks did not cover the whole wide-field illuminated area, FRAP photomasks of similar area were used for stimulation of 405-nm light at 9.92W/cm2 and 488 nm light at 41.92W/cm2.

Because channels with a stable open state are sensitive to broad-spectrum daylight conditions, channels were closed before and after each experiment by a brief illumination with the respective closing wavelength. Channelrhodopsins were characterized by their responses to 500-ms light pulses of 405, 488, 561, 594, and 639 nm, using the DL594 laser and no photomask for the 594 nm and the TIRF laser for the other wavelengths. To test focal optogenetic stimulation and record responses, 488-nm light pulses were delivered using DirectFRAP photomasks and the 100× objective; 594-nm Rapp Opto custom-designed photomasks were overlaid to produce a coillumination donut surrounding a central region.

Microscope and components including lasers were controlled with Zeiss AxioVision and Zen Blue software. Transfected cells were identified by fluorescence and selected based on health and membrane integrity. Recordings were acquired from cells voltage-clamped at −70mV. Liquid junction potential, pipette, and cell capacitance influences were compensated. Recordings were managed with IGOR Pro (Wavemetrics; version 6.22A) and the Patcher’s Power Tools extension for HEKA files provided by the Department of Membrane Biophysics at the Max Planck Institute for Biophysical Chemistry in Goettingen. In cases where small shifts in timing of laser pulses occurred, traces were manually aligned to illumination onset for comparison.