Patch-clamp electrophysiology

For voltage-clamp recordings, patch electrodes (3–5 MΩ) were back-filled with a cesium methanesulfonic acid solution containing (in mM): 117 Cs methanesulfonic acid, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEA, 2 ATP, 0.2 GTP. For current-clamp recordings, patch electrodes (3–5 MΩ) were back-filled with a potassium gluconate solution containing (in mM): 130 K-gluconate, 10 KCl, 10 HEPES, 10 EGTA, 2 MgCl₂, 2ATP, 0.2 GTP. pH = 7.35, 270–285 mOsm for all internal solutions. Cells were visualized using infrared differential contrast and fluorescence microscopy. Whole-cell recordings were made using a MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, California). For optical photostimulation, blue light (pE-4000, 460 nm, CoolLED, Andover, UK) was filtered with the EX-LED excitation filter (nVoke, Inscopix, Palo Alto, California) and delivered through a 40X objective. Orange light (pE-4000, 550 and 635 nm at a 100:15 ratio, CoolLED, Andover, UK) was filtered with the OG-LED excitation filter (nVoke, Inscopix, Palo Alto, California) and delivered through a 40X objective. Series resistance (15–25 Ω) and/or input resistance was monitored online using a 5 mV hyperpolarizing step delivered between stimulation sweeps. All data was filtered at 2 kHz, digitized at 5 kHz, and collecting using pClamp10 software (Molecular Devices, Sunnyvale, California).

For ChrimsonR synaptic terminal experiments, OG-LED light pulses (5 ms, 2 mW/mm²) or EX-LED light pulses (5 ms, 2 mW/mm²) were delivered every 20 s to evoke postsynaptic currents. EPSC amplitudes were calculated by measuring the peak current from the average response from >5 sweeps. For the ChrimsonR synaptic terminal timecourse experiment, OG-LED light pulses (5 ms pulses, 10 Hz, 13 mW/mm²) were delivered every 20 s for 5 sweeps to evoke postsynaptic currents. Pulse trains were repeated every 5 min for a total of 30 min of recording. In a subset of neurons, EX-LED (20 min, 0.5 mW/mm²) was delivered between minutes 5 and 25. EPSC amplitudes were calculated by measuring the peak current from the average response from 5 sweeps. In a few cases, BLA terminal stimulation elicited spikes. As necessary, EPSC amplitude was estimated by generating an exponential curve fit to the latter 300–400 ms of the mean EPSC trace. EPSC amplitude was estimated as the intercept point of the extrapolated curve fit and the rising phase of the EPSC trace.

For all somatic recordings, OG-LED light pulses (500 ms, 0.2–2.0 mW/mm²) or EX-LED (500 ms, 0.2–2.0 mW/mm²) were delivered every 20 s to evoke postsynaptic currents, or action potential firing. EPSC amplitudes were calculated by measuring the peak current from the average response from 5 sweeps.