Multiclamp 700b amplifier

Recordings were made using a Multiclamp 700B amplifier (Axon Instruments), filtered at 3 kHz and sampled at 10 kHz. Data analysis was performed with custom software written in MatLab. Experiments were discarded if the holding current was less than −600 pA or if the series resistance was greater than 25 MΩ. Series resistance across simultaneously recorded cells was within 25% for each pair. Recordings were performed at room temperature (19–21 °C). The amplitude of IPSCs was calculated by averaging the amplitude 0.5 ms before to 2 ms after the peak of the current. The unsigned magnitude of synaptic currents are shown for clarity.

Once cultured and transfected, we whole-cell voltage-clamped green-fluorescent Chronos-expressing CHO cells using a Multiclamp 700B amplifier (Axon Instruments, Molecular Devices). Membrane currents were digitised with a Power1401 (Cambridge Electronic Devices) controlled by Spike2 software (V5, Cambridge Electronic Devices).
Coverslips with adhered transfected cells were bathed at room temperature in a solution containing (in mM [45 (link)]): 140 NaCl, 5 KCl, 10 CaCl₂, 2 MgCl₂, 0.3 Na₂HPO₄, 0.4 KH₂PO₄, 4 NaHCO₃, 5 glucose, 10 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES). The osmolality of the solution was adjusted to 300–310 mOsmol kg⁻¹ with a 1M sucrose, and the pH was adjusted to 7.3  ±  0.01 with a 1M NaOH. The patch pipette was filled with an artificial intracellular solution containing (in mM [45 (link)]): 150 K-gluconate, 2 MgCl₂, 1.1 ethylene glycol-bis(2-aminoethylether)-N,N, ${\mathrm{N}}^{\prime }$ , ${\mathrm{N}}^{\prime }$ -tetraacetic acid (EGTA), 5 HEPES. The osmolality was adjusted to 290 mOsmol kg⁻¹, and the pH was adjusted to 7.3  ±  0.01 with 1M KOH.
Patch pipettes were pulled from glass capillaries (PG10150-4, World Precision Instruments) to tips with resistances between 4 and 8 MΩ. Voltage was clamped at  −40 mV. Cells with leak currents of a magnitude greater than 100 pA were excluded from the analysis. We monitored the access and input resistances between photostimulation epochs.

Glass pipettes (Hilgenberg) were filled with internal solution containing in mM: K-gluconate 140, MgCl₂ 1, NaCl 8, Na₂ATP 2, Na₃GTP 0.5, HEPES 10, Tris-phosphocreatine 10 to pH 7.2 with KOH (all by Sigma-Aldrich). For electrophysiological recordings of L2/3 pyramidal cells, pipette with resistance of 3–6 MΩ were used, whereas for L4 recordings the pipette resistance was in the range of 7–14 MΩ. Cells depth within the tissue was inferred from the position of the glass pipette with respect to the pial surface. The range of depths was 110–380 μm for L2/3 cells and 410–500 for L4 cells. For experiments in Figures 3 and 4 and Supplementary Figures 2–4, 20–30 consecutive acquisitions (trials, acquisition duration: 4–8 s) were performed for each experimental condition and for experiments in Figures 5 and 6 and Supplementary Figures 5 and 6, for each stimulus direction. For experiments in Figure 2, 12–30 trials, for each experimental condition, were acquired. Data were collected through a Multiclamp 700B amplifier, sampled at 50 kHz, and filtered at 10 kHz by a Digidata 1440 acquisition system (Axon Instruments).