Optogenetic silencing strategies differ in their effects on synaptically-evoked spiking activity. (a) Top left, confocal image of a CA3 pyramidal neuron expressing eNpHR3.0-EYFP (‘NpHR’). Bottom, cell-attached recordings from this cell showing synaptically-evoked spiking before (left) and after (right) NpHR-activation (15 s, 532 nm, 7.9 mW mm⁻²). Spike probability was set to approximately 0.4 before laser-activation (measured over 10 trials). The ‘before’ stimulus was delivered 1250 ms before laser onset and the ‘after’ stimulus was delivered 250 ms after laser offset. Top right, summary of spike probability for NpHR cells (n = 10, error bars indicate s.e.m.). (b) Top left, a CA3 pyramidal neuron expressing Arch-GFP (‘Arch’). Bottom, cell-attached recordings from this cell showing synaptically-evoked spiking before (left) and after (right) Arch-activation (15 s, 532 nm, 76.1 mW mm⁻²). Top right, summary of spike probability for Arch cells (n = 12). All conventions as in ‘a’ (c) Perforated patch current clamp recording from a NpHR-expressing neuron. Laser-activation (10.9 mW mm⁻² laser for 15 s) evoked a sustained hyperpolarizing response (left). In the absence of laser-activation, GABA puffs elicited hyperpolarizing responses (middle). However, the same GABA puff generated a depolarizing response and action potential when delivered 250 ms after laser-activation (right).

A light-activated Cl⁻ pump, but not a H⁺ pump, causes a sustained change in GABAergic transmission. (a) Top, gramicidin perforated patch voltage clamp recording from a neuron expressing eNpHR3.0-EYFP. GABA_AR currents were measured before and after NpHR-activation using three different laser intensities: ‘zero’ (light grey), ‘intermediate’ (dark grey) and ‘higher’ (black). Note the change in direction of current flow through the GABA_AR as a function of NpHR-activation. Bottom, laser intensities were selected by assessing their effectiveness in silencing spikes evoked by somatic current injection in current clamp (see Supplementary Information). (b) Recordings from a neuron expressing Arch-GFP. All conventions as in ‘a’. Note the consistent GABA_AR current for different levels of Arch-activation. (c) Estimating the effects of photocurrents on E_GABAA for the NpHR cell in ‘a’ (top) and Arch cell in ‘b’ (bottom). GABA_AR IV plots (left) were used to calculate the resting E_GABAA and GABA_AR conductance (gGABAA), which were then used to estimate E_GABAA for individual GABA puffs delivered after different mean photocurrents (right; symbol colours correspond to data in ‘a’ and ‘b’). (d) Summary of the change in E_GABAA associated with different NpHR (black symbols) and Arch (grey symbols) photocurrents. Small empty symbols indicate data from organotypic hippocampal slices. Small filled symbols indicate data from acute hippocampal slices. Large symbols with error bars (s.e.m.) indicate combined population averages. e) Traces from a representative NpHR-expressing neuron showing GABA_AR currents recorded at different times after the photocurrent, on different trials. E_GABAA versus time after photocurrent is plotted for this cell and the recovery is well fitted by a single-exponential function. Inset plot shows the distribution of time constants of E_GABAA recovery for all NpHR-expressing cells. (f) Traces from a representative NpHR-expressing neuron showing GABA_AR currents recorded after photocurrents of different durations. Plot illustrates the change in E_GABAA as a function of photocurrent duration for all NpHR-expressing cells.