Figure 4. Extent and specificity of light-activation of opsin-expressing cells.
(a) Diagram of illumination. The fibres used in this study have a numerical aperture (NA) of 0.37 and a radius (r) of 100 μm. With an index of refraction (n) for brain tissue of 1.35, and a flat cleave tip, there is a half-angle (θ) of ~16°. Given our average power measured post hoc from the implanted fibre tip, and reported irradiance values for activating opsins, we reach a depth (d) of 0.55 mm for HR with amber light and 0.9 mm for ChR2 with blue light. Note that the diagram is not drawn to scale. (b) c-Fos expression is not induced in PV cells after 2 h of on-demand light (green diamonds), but can be induced with continuously pulsed blue light for the entire 2 h (that is, not on-demand light application, red squares) in PV-ChR2 opsin-expressing animals. Note that this c-Fos expression induced by 2 h long pulsed light application reflects both direct and indirect activation over a prolonged time period (for example, through gap-junctions and network connexions). An increase in PV-cell c-Fos expression was not observed without opsin expression (grey triangles). Light was delivered, and c-Fos expression examined, contralateral to the site of KA injections. (c) Example PV cells from PV-ChR2 opsin-expressing (top) and opsin-negative (bottom) animals in b. Green: PV. Red: c-Fos. Yellow: co-localization. Scale bar, 10 μm. (d,e) Long pulses of light (2 s on, 50 ms off; orange bars) produced robust outward (inhibitory) currents in CA1 pyramidal (d) and granule cells (e) of epileptic Cam-HR opsin-expressing animals (green traces), but not epileptic opsin-negative littermate controls (black traces). Scale bars, 1 s, 100 pA. Holding potential, −60 mV. (f) A single pulse of light (10 ms) produces robust postsynaptic currents in two CA1 pyramidal cells (dual recording) from a KA-treated PV-ChR2 opsin-expressing animal (green bolded trace, average of 15 individual sweeps, shown in light green). No-light-induced responses were seen in opsin-negative animals (bottom, average trace shown in black). Scale bars, 20 ms, 40 pA. Holding potential, −80 mV.