Investigating the feasibility of channelrhodopsin variants for nanoscale optogenetics

Abstract

Optogenetics has revolutionized the study of circuit function in the brain, by allowing activation of specific ensembles of neurons by light. However, this technique has not yet been exploited extensively at the subcellular level. Here, we test the feasibility of a focal stimulation approach using stimulated emission depletion/reversible saturable optical fluorescence transitions-like illumination, whereby switchable light-gated channels are focally activated by a laser beam of one wavelength and deactivated by an overlapping donut-shaped beam of a different wavelength, confining activation to a center focal region. This method requires that activated channelrhodopsins are inactivated by overlapping illumination of a distinct wavelength and that photocurrents are large enough to be detected at the nanoscale. In tests of current optogenetic tools, we found that ChR2 C128A/H134R/T159C and CoChR C108S and C108S/D136A-activated with 405-nm light and inactivated by coillumination with 594-nm light-and C1V1 E122T/C167S-activated by 561-nm light and inactivated by 405-nm light-were most promising in terms of highest photocurrents and efficient inactivation with coillumination. Although further engineering of step-function channelrhodopsin variants with higher photoconductances will be required to employ this approach at the nanoscale, our findings provide a framework to guide future development of this technique.

Keywords: RESOLFT microscopy; STED microscopy; channelrhodopsin; optogenetics.

Fig. 1

Channelrhodopsin localization and diffusion in the membrane. (a) STED microscopy image of ChR2-EYFP in the dendrites of a transfected hippocampal neuron in culture. $\text{scale bar}=300\mathrm{nm}$. (b) FRAP experiments of ChR2-EYFP in transfected neuronal processes (top) and cell bodies (bottom) before (left), immediately after (middle), and 200 s after (right) photobleaching of the indicated area. $\text{scale bars}=20\mu \mathrm{m}$. R1 and R2 indicate nonbleached control regions, and R3 indicates background fluorescence black level, used for normalization of fluorescence recovery following photobleaching. (c) Quantitation of FRAP in neuronal processes (top; $n=42$) and cell bodies (bottom; $n=48$). Error bars indicate SEM.

Fig. 2

Illumination evoked photocurrents of light-gated channels with fast photocycles. (a) Photocurrents from recorded hippocampal neurons in culture expressing H134R, (b) T159C, (c) E123T/T159C, and (d) L132C channels, elicited by 405, 488, 561, 594, and 639 nm single wavelength laser illumination (top panels) and by coillumination with different wavelengths (lower panels). Photocurrent recordings at specific illumination wavelengths are indicated by the respective color of the recording. Scale bars above current traces indicate recorded current (vertical) and time (horizontal). For coillumination experiments, bars below recordings indicate time of illumination for the indicated color-coded wavelength. Black bars refer to coillumination with the color of the depicted traces in cases where more than one coillumination wavelength was tested. Activating laser intensities were 405 nm, $87\mathrm{mW}/{\mathrm{cm}}^2$; 488 nm, $296\mathrm{mW}/{\mathrm{cm}}^2$; 561 nm, $146\mathrm{mW}/{\mathrm{cm}}^2$; and 639 nm, $22\mathrm{mW}/{\mathrm{cm}}^2$, unless otherwise indicated.

Fig. 3

Illumination evoked photocurrents of redshifted light-gated channels. (a)–(d) Photocurrents of the indicated channels elicited by 405, 488, 561, 594, and 639 nm single wavelength laser illumination (top panels) and by coillumination with different wavelengths (lower panels). Photocurrent recordings at specific illumination wavelengths are indicated by the respective color of the recording. Scale bars above individual experiments indicate recorded current (vertical) and time (horizontal). For coillumination experiments, bars below recordings indicate time of illumination for the indicated color-coded wavelength. Black bars refer to coillumination with the color of the depicted traces in cases where more than one coillumination wavelength was tested. Activating laser intensities were 405 nm, $87\mathrm{mW}/{\mathrm{cm}}^2$; 488 nm, $296\mathrm{mW}/{\mathrm{cm}}^2$; 561 nm, $146\mathrm{mW}/{\mathrm{cm}}^2$; and 639 nm, $22\mathrm{mW}/{\mathrm{cm}}^2$, and inactivating laser intensities were 405 nm, $662\mathrm{mW}/{\mathrm{cm}}^2$; 488 nm, $3.15\mathrm{W}/{\mathrm{cm}}^2$; 561 nm, $1.33\mathrm{W}/{\mathrm{cm}}^2$; 594 nm, $801\mathrm{mW}/{\mathrm{cm}}^2$; and 639 nm, $283\mathrm{mW}/{\mathrm{cm}}^2$.

Fig. 4

Illumination evoked responses of light-gated ion-channels with slow photocycles (step-function opsins). (a) Responses to 405, 488, 561, 594, and 639 nm single wavelength laser illumination and coillumination of C128S/D156A (step-function opsin), (b) C128/H134R/T159C, and (c) C128S/L132C/T159C switchable channels. Photocurrent recordings at specific wavelength illumination are indicated by the respective color of the recording. Scale bars above individual experiments indicate current (vertical) and time (horizontal). For coillumination experiments, bars below recordings indicate time of illumination for the indicated color-coded wavelength. Black bars refer to coillumination with the color of the depicted traces in cases where more than one coillumination wavelength was tested. Activating laser intensities were 405 nm, $87\mathrm{mW}/{\mathrm{cm}}^2$; 488 nm, $296\mathrm{mW}/{\mathrm{cm}}^2$; 561, $146\mathrm{mW}/{\mathrm{cm}}^2$; 594 nm, $80\mathrm{mW}/{\mathrm{cm}}^2$; and 639 nm, $22\mathrm{mW}/{\mathrm{cm}}^2$, unless otherwise indicated.

Fig. 5

Illumination evoked responses of redshifted and higher photocurrent light-gated ion-channels with step-function mutations. (a) Responses to 405, 488, 561, 594, and 639 nm single wavelength laser illumination and coillumination of (a) C1V1 E122T/C167S, (b) CoChR C108S, and (c) CoChR C108S/D136A channels. Photocurrent recordings at specific wavelength illumination are indicated by the respective color of the recording. Scale bars above individual experiments indicate current (vertical) and time (horizontal). For coillumination experiments, bars below recordings indicate time of illumination for the indicated color-coded wavelength. Black bars refer to coillumination with the color of the depicted traces in cases where more than one coillumination wavelength was tested. Activating laser intensities were 405 nm, $87\mathrm{mW}/{\mathrm{cm}}^2$; 488 nm, $296\mathrm{mW}/{\mathrm{cm}}^2$; 561 nm, $146\mathrm{mW}/{\mathrm{cm}}^2$; 594 nm, $80\mathrm{mW}/{\mathrm{cm}}^2$; and 639 nm, $22\mathrm{mW}/{\mathrm{cm}}^2$, and inactivating laser intensities were 405 nm, $662\mathrm{mW}/{\mathrm{cm}}^2$; 488 nm, $3.15\mathrm{W}/{\mathrm{cm}}^2$; 561 nm, $1.33\mathrm{W}/{\mathrm{cm}}^2$; 594 nm, $801\mathrm{mW}/{\mathrm{cm}}^2$; and 639 nm, $283\mathrm{mW}/{\mathrm{cm}}^2$, unless otherwise indicated.

Fig. 6

Illumination evoked responses of equimolar ChR2 H134R and NpHR3.0 (eNPAC). Evoked responses of eNPAC with 405, 488, 561, 594, and 639 nm single wavelength laser (a) illumination and (b) coillumination. Scale bars above individual experiments indicate current (vertical) and time (horizontal). For coillumination experiments, bars below recordings indicate time of illumination for the indicated color-coded wavelength. Black bars refer to coillumination with the color of the depicted traces. Activating laser intensities were 405 nm, $87\mathrm{mW}/{\mathrm{cm}}^2$; 488 nm, $296\mathrm{mW}/{\mathrm{cm}}^2$; and 561 nm, $146\mathrm{mW}/{\mathrm{cm}}^2$, and inactivating 594 nm coillumination intensity was $801\mathrm{mW}/{\mathrm{cm}}^2$.

Fig. 7

Reduction of 488- and 405-nm evoked photocurrents from ChR2 C128A/H134R/T159C, CoChR C108S, and CoChR C108S/D136A channels. (a) Effect of coillumination of different 594-nm laser powers on 488 nm (top) or 405 nm (bottom) evoked currents; 594-nm laser powers are indicated in the color-coded inset and were tested in 10% steps from 0% to 100% laser power. (b) Relationship between 594-nm laser intensity and photocurrent reduction. (c) Influence of activating 488 nm (top) or 405 nm (bottom) laser power on 594 nm light-mediated photocurrent reduction. Scale bars indicate recorded currents (vertical) and time (horizontal). (d) Photocurrent reduction of 488-nm light (top) and 405-nm light (bottom) evoked currents in CoChR C108S (left) and C108S/D136A (right) mutants. Trace and stimulation bar colors represent laser wavelengths and intensities (5%, 10%, 15%, and 20%).

Fig. 8

Summary of activating/inactivating wavelengths, photocurrent magnitude, and degree of inactivation for all channels tested. (a) Magnitude of photocurrent (blue) and portion of photocurrent inactivated (orange) by the indicated activation/inactivation intensities ($\mathrm{mW}/{\mathrm{cm}}^2$) color-coded by wavelength, for all channels tested, and for selected candidates (b).

Fig. 9

Electrophysiological recordings of photocurrents in focally stimulated areas. (a) T159C channelrhodopsin photocurrents elicited by $90.48\mathrm{W}/{\mathrm{cm}}^2$ 488 nm full field (left) or focal (right) activation of the cell body. (b) Illumination scheme for focal activation based on STED/RESOLFT microscopy: 488-nm activation light is brought together with a 594-nm donut-shaped inactivation beam. The two wavelengths of illumination overlap and inactivate channels within the donut region, resulting in a potentially subdiffraction limited central activated region. (c) Images of yellow text highlighter, used as a dye for a uniform fluorescent field on a coverslip illuminated with 488-nm light in a $16.7\text{-}\mu \mathrm{m}$ diameter central region, and 594-nm light in an overlapping donut region, resulting in a central activated region of $6.2\mu \mathrm{m}$. $\text{scale bar}=10\mu \mathrm{m}$. (d) eNPAC photocurrents elicited by 488 nm light in a focal $6.2\mu \mathrm{m}$ (blue) or $16.7\mu \mathrm{m}$ (black) diameter region, or in a $16.7\text{-}\mu \mathrm{m}$ region combined with a 594-nm laser overlapping donut to inactivate the overlapping region resulting in a central activated region of $6.2\mu \mathrm{m}$ (black trace). 594-nm laser intensity can be tuned to drive NpHR3.0-mediated hyperpolarizing currents to be weaker (orange trace), stronger (red trace), or equivalent to (black trace) the photocurrent elicited by $6.2\mu \mathrm{m}$ stimulation of ChR2 (blue trace). (e) Photocurrents of the ChR2 C128S/H134R/T159C variant evoked by 6.2 or $16.7\mu \mathrm{m}$ regions of $9.92\mathrm{W}/{\mathrm{cm}}^2$ 405-nm laser illumination and subsequent 594-nm illumination mediated closure (left), and with coillumination of $16.7\mu \mathrm{m}$ 488-nm illuminated region and an overlapping donut of 594-nm illumination, resulting in a $6.2\text{-}\mu \mathrm{m}$ central activated region (right). When a $16.7\text{-}\mu \mathrm{m}$ 488-nm illuminated region was combined with a 594-nm overlapping donut region (leaving a central $6.2\mu \mathrm{m}$ region of activation), the photocurrent is reduced to a level similar to that evoked by a $6.2\text{-}\mu \mathrm{m}$ region alone.