Brain slice recordings

Brain slices of the left hemisphere containing V1 and SC were prepared 3 to 4 weeks after viral injection of ChR2. Mice (age 7 to 8 weeks) were deeply anesthetized with isoflurane (3% in oxygen) and immediately decapitated. The brain was rapidly extracted and immersed in an ice-cold cutting solution of composition: 100 mM Choline Chloride, 11.60 mM N-ascorbate, 7 mM MgCl₂, 3.10 mM Na-pyruvate, 2.50 mM NaH₂PO₄, 0.50 mM CaCl₂ (pH = 7.4). Brain slices (300 μm thick) containing the left V1 and SC were cut in a coronal plane using a Leica Vibratome 1000S. Slices were transferred to warm (35°C) incubating solution of composition: (92 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 30 mM NaHCO₃, 20 mM HEPES, 25 mM glucose, 3 mM sodium pyruvate, 2 mM MgSO₄, 2 mM CaCl₂) for 30 min, which was then allowed to gradually cool to room temperature for another 30 min before being used for recording.

During recording slices were continuously perfused with oxygenated, heated (34°C) artificial cerebral spinal fluid (ACSF) of composition: 125 mM NaCl, 25 mM NaHCO₃, 3 mM KCl, 1.25 NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂ and 25 mM glucose (pH = 7.4; bubbled with 95% O₂ / 5% CO₂). The region of SC containing V1 projections was identified by the presence of green fluorescent. Whole-cell somatic recordings were performed under visual control using a 60x objective on an Olympus BX50 microscopy equipped with differential interference contract optics and infrared illumination (Stuart et al., 1993). Patch pipettes with an open tip impedance of 5 to 7 MΩ were filled with an internal solution containing: 10 mM KCl, 130 mM K-Gluconate, 10 mM HEPES, 4 mM Mg²⁺ATP, 0.3 mM Na₂GTP, 10 mM Na₂Phosphocreatine and 0.5% biocytin (pH = 7.25; KOH). All recordings were made in current-clamp mode using a current clamp amplifier (Dagan BVC-700A). To characterize electrophysiological properties of SC neurons, hyperpolarizing and depolarizing current steps of 1,000 ms duration (from −400 pA to +600 pA, 50 pA/step) were applied via the somatic recording pipette. Voltage signals were filtered at 10 kHz and digitized at 20 or 50 kHz using an A-D converter (ITC-18, Instrutech/HEKA, Germany) under the control of Axograph acquisition software (Axograph X, Axograph Scientific, Sydney, Australia) running on an Apple Macintosh computer (iMAC-i7). Axograph was used for both data acquisition and primary analysis.

Full-field optogenetic stimulation was performed with 470 nm blue light pulses (2 ms duration) via an LED light source (ThorLab, United States) attached to the epifluorescence port of the microscope. A GFP (green fluorescent protein) filter cube (Chroma) was inserted into the light path to direct 470 nm LED light onto the brain slice. For each neuron we averaged the response to 10 trails of LED stimulation at different intensities (0.7 mW, 1.4 mW, 1.9 mW, 2.5 mW, 2.9 mW and 4.8 mW).

In some experiments, one or more the following pharmacological agents was added to the ACSF perfusing the slice: The sodium-channel blocker tetrodotoxin (TTX; 1 μM; Hello Bio), the potassium-channel blocker 4-aminopyridine (4-AP, 100 μM, Sigma), the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 μM; Tocris) and the N-methyl-D-aspartate (NMDA) receptor blocker amino-5-phosphonovaleric acid (APV; 25 μM; Tocris).